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Hepatic SIRT6 protects against cholestatic liver disease primarily via inhibiting bile acid synthesis
Hongjun Peng, Department of Pediatrics, Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University, 321 Zhongshan Road, Nanjing, Jiangsu 210008, China. E-mail: hjpeng@njglyy.com
Longfeng Jiang, Department of Infectious Diseases, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu 210029, China. E-mail: longfengjiang@njmu.edu.cn
Liang Sheng, Department of Pharmacology, School of Basic Medical Science, Nanjing Medical University, 101 Longmian Avenue, Nanjing, Jiangsu 211166, China. E-mail: lgsheng@njmu.edu.cn
Cholestatic liver disease, caused by the accumulation of hazardous bile acids in the liver, may result in cirrhosis, fibrosis, or liver failure. Activation of SIRT6 prevents cholestasis-associated pathological events, such as oxidative stress and mitochondrial biogenesis disorders, and inhibits bile acid synthesis to alleviate cholestatic liver injury. However, it is still uncertain which pathway is responsible for the therapeutic effect of SIRT6 in reducing cholestasis. Therefore, we treated liver-specific Sirt6 knockout mice with N-Acetylcysteine, Keap1-Nrf2-IN-1, or acadesine to remove oxidative stress and/or trigger mitochondrial biogenesis after cholestatic liver disease modeling, but these measures did not significantly improve cholestatic symptoms. However, MDL801, a SIRT6 agonist that downregulating CYP7A1, the key enzyme in bile acid synthesis, exhibited favorable therapeutic effects. In addition, the hepatic knockdown of Cyp7A1 further confirmed that inhibition of hepatic bile acid synthesis might be the main pathway by which SIRT6 alleviates cholestatic liver disease. These findings provide a solid basis for the potential application of SIRT6 agonists in the treatment of cholestatic liver disease.
Cholestasis is a disorder that causes bile stasis and the accumulation of hazardous bile acids in the liver and bloodstream. Cholestasis may arise from genetic and developmental abnormalities such as biliary atresia, as well as acquired diseases like those caused by viral hepatitis, drugs, metabolic syndrome, primary sclerosing cholangitis, primary biliary cholangitis, alcoholic liver disease, and obstruction by tumors or gallstones[1].
Bile acids, particularly those with hydrophobic properties, may be highly destructive to cells. Bile acids invade cellular mitochondria, inhibit the activity of the mitochondrial respiratory chain complex, and cause an obstacle to the electron transfer in the respiratory chain. On the one hand, it increases the production of reactive oxygen species in hepatocytes and induces oxidative stress; on the other hand, it leads to ATP depletion, disrupts mitochondrial membrane potential, increases mitochondrial permeability, and induces cell death. In addition, mitochondria have their DNA (mtDNA) encoding 13 key subunits of the respiratory chain complex, which is highly susceptible to damage by oxidative stress. mtDNA damage leads to impaired expression of the respiratory chain complex, further exacerbating oxidative stress and mitochondrial dysfunction[2–6]. The persistence of oxidative stress and mitochondrial dysfunction activates inflammatory signaling pathways within hepatocytes, leading to the release of cytokines and chemokines that further recruit immune cells to enter the liver and create a chronic inflammatory state[7]. Inflammation and cellular damage activate hepatic stellate cells and promote collagen deposition, leading to alterations in liver structure and further deterioration of liver function[8]. Therefore, without timely treatment, chronic cholestasis may result in cirrhosis, fibrosis, and ultimately liver failure requiring a liver transplant[9].
Sirtuin 6 (SIRT6), a member of the sirtuin family, possesses nicotinamide adenine dinucleotide‐dependent activity of deacetylase, mono-adenosine diphosphate ribose-ribosyltransferase, and deacylating long-chain fatty acids[10–11], and controls a variety of biological functions, including oxidative stress, inflammation, and mitochondrial biology[12] that are involved in the development of cholestatic liver disease. It has been reported that SIRT6 counteracts oxidative stress by activating nuclear factor erythroid 2-related factor 2 (NRF2) that mediates the transcription of a range of antioxidant genes[13–16]. SIRT6 also activates AMP-activated protein kinase (AMPK), which in turn activates the transcriptional activity of peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α) through phosphorylation modification and enhances the action of PGC1α on its own promoter, thereby accelerating the expression of PGC1α. As a regulator of mitochondrial biosynthesis, PGC1α enables SIRT6 to upregulate mitochondrial number and promote mitochondrial function[17–19]. In addition, SIRT6 inhibits the ERRγ-induced transcription of CYP7A1, a key enzyme for bile acid synthesis, through deacetylation of estrogen-related receptor γ (ERRγ), thereby potentially attenuating bile acid accumulation and cholestasis[20].
To investigate which pathway is responsible for the therapeutic effect of SIRT6 in reducing cholestasis, we used N-acetylcysteine (NAC), Keap1-Nrf2-IN-1 (KNI-1), acadesine (AICAR), or adeno-associated virus knocking down CYP7A1 to treat hepatic Sirt6-deficient mice. NAC, a synthetic derivative of L-cysteine, acts as a precursor for glutathione synthesis and enhances the activities of superoxide dismutase, glutathione reductase, and glutathione peroxidase in hepatocytes by up-regulating the supplemental glutathione level to resist oxidative stress[21]. KNI-1 disrupts the interaction between NRF2 and its inhibitory binding protein, Keap1, thereby preventing the degradation of NRF2, activating the expression of oxidative genes downstream of NRF2, and protecting the cells from oxidative stress[22]. AICAR, an activator of AMPK, promotes mitochondrial biosynthesis by upregulating PGC1α expression[23].
The current study aims to elucidate the molecular mechanism by which SIRT6 alleviates cholestasis. We employed active small molecules and gene knock-down techniques to examine how SIRT6 might influence cholestasis, specifically focusing on its role in managing oxidative stress, promoting mitochondrial biosynthesis, and inhibiting bile acid synthesis, in mice with hepatocyte-specific Sirt6 deletion.
Materials and methods
Animals
All practices concerning animal husbandry and experimentation followed the applicable institutional and national guidelines, and were officially approved by the Animal Care and Ethical Committee of Nanjing Medical University (Approval No. 1811047). Jackson Laboratory in Maine, USA, provided the mice with Sirt6 exons flanked by loxP sites (Sirt6f/f) and Albumin-Cre transgenic mice on a C57BL/6 background. To establish a hepatocyte-specific deficiency in Sirt6 (Sirt6Δhep), Sirt6f/f mice were bred with Albumin-Cre mice as reported previously[24]; Sirt6f/f mice were used as the wild-type counterparts. Wild-type C57BL/6 mice were obtained from the Animal Core Facility based at Nanjing Medical University in Nanjing, China.
Seven-week-old male mice were infected with either adenovirus carring Sirt6 (Ad-Sirt6) or control adenoviruses (Ad-Control) at a dose of 5 × 1010 viral particles per mouse through tail vein injection, and surgery was conducted seven days afterward.
Five-week-old male mice were infected with adeno-associated virus delivering shCyp7A1 or control adeno-associated virus (shControl) at a dosage of 2 × 1011 genomic copies (GC) per mouse through tail vein injection, and surgery was conducted 21 days afterward.
Male mice at the age of eight weeks were anesthetized before undergoing either sham surgery or bile duct ligation (BDL)[25]. Briefly, a midline laparotomy was performed, and then the common bile duct was double-ligated with 4-0 suture and transected between ligations. The mice in the sham group received similar surgery except for the bile duct ligation and transection. After surgery, the mice were placed on a heating pad in a cage and heated with an infrared lamp until the mice were fully awake and active.
The survival rate was monitored for 22 days following the surgical procedure. Blood samples and liver tissues gathered six days post-surgery will be preserved at −80 ℃.
The timing of all mouse treatments is shown in Supplementary Fig. 1 (available online).
Drug administration
MDL801 [100 mg/(kg·day); TargetMol, Shanghai, China], KIN-1 (40 mg/(kg·day); MCE, Shanghai, China), NAC [200 mg/(kg·day); Solarbio, Beijing, China], and AICAR [250 mg/(kg·day); MCE, Shanghai, China) were administered to mice intraperitoneally one day before BDL surgery, and the administration continued once daily after BDL for another six times. The timing of all mouse treatments is shown in Supplementary Fig. 1 (available online).
Packaging and purification of virus
Adeno-associated virus serotype 8 (AAV8) delivering shCyp7a1 and shControl were prepared by Applied Biological Materials Inc. (Richmond, Canada). The titers of shCyp7a1 and shControl viruses were 1.54 × 1012 genomic copies (GC)/mL and 1.95×1012 GC/mL, respectively. The Ad-Sirt6 and Ad-Control were purchased from Hanbio, Shanghai, China. The virus titers of Ad-Sirt6 and Ad-Control were 1.07 × 1012 viral particles (VP)/mL and 1.34×1012 VP/mL, respectively.
Hematoxylin-eosin (H&E) staining
Liver tissue were immersed in 4% paraformaldehyde for 48 hours, embedded in paraffin, , and carefully sectioned at 6 μm. The tissue samples underwent H&E staining, using a staining kit from Beyotime Biotechnology in Shanghai, China. Representative images were captured using Leica microsystems (Solms, Germany). H&E staining images were quantified by Image J software to determine necrosis area.
Determination of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and bile acid levels
We examined the activities of ALT and ALP in the blood to assess liver injury. Blood samples were collected and spun at 1200g for 10 minutes to obtain serum. Serum ALT and ALP activities were measured using commercially available assay kits according to the manufacturer's instructions (C009-2-1 and C010-2-1, Jiancheng, Nanjing, Jiangsu, China). Serum total bile acid levels in serum were measured using a bile acid colorimetric assay kit (E-BC-K181-M, Elabscience, Wuhan, China) following the manufacturer's instructions.
Measurement of malonaldehyde (MDA), ATP, ROS, and cytochrome C (Cyt C) contents
Liver tissues were rinsed with PBS and then homogenized and sonicated in lysis buffer on ice. After sonication, the lysed tissues were centrifuged at 10000g for 10 min to remove debris to eliminate any debris. The MDA and ATP content of the liver was measured with MDA (Cat. #BC0025, Solarbio, Beijing, China) and ATP assay kit (Cat. #BC0305, Solarbio, Beijing, China). The hepatic ROS levels were determined using a ROS kit (Cat. #E004-1-1, Jiancheng, Nanjing, Jiangsu, China) according to the manufacturer's instructions. Cytoplasmic Cyt C content in the liver was assayed by an enzyme-linked immunosorbent assay kit (Cat. #ab210575, Abcam, Minneapolis, MN, USA) and normalized to total liver protein. Liver cytosol was isolated following the instructions of this kit.
Immunoblotting
Total proteins from liver tissues were extracted according to our laboratory approach described previously[24], separated by SDS-PAGE, immunoblotted with the indicated primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (1∶5000, anti-mouse IgG, Cat. #6805020 and anti-rabbit IgG, Cat. #671280, Biosharp, Hefei, Anhui, China), and finally visualized with chemiluminescent horseradish peroxidase substrate (Cat. #1619502, Millipore, Boston, MA, USA) using a Tanon-5200 Chemiluminescence Imager (Tanon, Shanghai, China). Antibodies against SIRT6 (1∶1000; Cat. #12486), FLAG(1∶5000; Cat. #2368), BAX (1∶1000; Cat. #2772), cleaved CASPASE-3 (1∶1000; Cat. #9664s), H3 (1∶1000; Cat. #4499), AC-H3K9 (1∶1000; Cat. #9649), AMPKα (1∶1000; Cat. #5831), p-AMPKα (Thr172) (1∶1000; Cat. #2535) were purchased from Cell Signaling Technology (Danvers, MA, USA), NRF2 (1∶1000; Cat. #16396-1-AP) and TUBULIN (1∶1000; Cat. #10068-1-AP) from Proteintech (Wuhan, Hubei, China), CYP7A1 (1∶1000; Cat. #sc-518007) from Santa Cruz Biotechnology (Los Angeles, CA, USA), and PGC1α (1∶1000; Cat. #ab54481) from Abcam (Minneapolis, MN, USA).
Quantitative reverse transcription-PCR (qRT-PCR)
Total RNAs of hepatocytes and livers were extracted by Trizol Reagent (Takara, Shiga, Japan). The StepOnePlus™ Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA) was utilized to conduct qRT-PCR, using Thunderbird SYBR Master Mix (Toyobo, Osaka, Japan). Primer sequences were as follows: Transcription factor A, mitochondrial (Tfam), 5′-ATTCCGAAGTGTTTTTCCAGCA-3′ (forward), 5′-TCTGAAAGTTTTGCATCTGGGT-3′ (reverse); heme oxygenase 1 (Ho1), 5′- AAGCCGAGAATGCTGAGTTCA-3 (forward)′, 5′-GCCGTGTAGATATGGTACAAGG A-3′ (reverse); isocitrate dehydrogenase (NADP+) 2 (Idh2), 5′-GGAGAAGCCGGTAGTGGAGA T-3′ (forward), 5′- GGTCTGGTCACGGTTTGGAA-3′ (reverse); Nad(p)h quinone dehydrogenase 1 (Nqo1), 5′-AGGATGGGAGGTACTCGAATC-3′ (forward), 5′-AGGCGTCCTTCCTTATATGCTA-3′ (reverse); 36B4, 5′-GGTCTGGTCACGGTTTGGAA-3′ (forward) and 5′-CCGCAGGGGCAGCAGTGGT-3′ (reverse).
Measurement of mtDNA
We extracted mtDNA from liver tissue using QIAamp DNA Mini kit (Qiagen in Valencia, CA, USA), and performed qRT-PCR according to the previous report[26]. The 36B4 gene was used as a marker for nuclear DNA, and the cytochrome C oxidase 1 (Cox1) gene was used for mtDNA. Primer sequences were as follows: Cox1, 5′-TCTACTATTCGG AGCCTGAGC-3′ (forward) and 5′-CAAAAGCATGGGCAGTTACG-3′ (reverse); 36B4, 5′-CGACCTGGAAGTCCAACTAC-3′ (forward) and 5′-ATCTGCTGCATCTGCTTG-3′ (reverse).
Hepatocyte isolation and culture
Hepatocytes were isolated from mice receiving BDL or sham surgery according to a previous protocol[27]. Briefly, mouse liver was perfused by Hank's balanced salt solution (HBSS) supplemented with 4 mM NaOH, 10 mM HEPES and 0.5 mM EGTA followed by digestion with collagenase II solution (Cat. #LS004177, Worthington Biochemical Corporation, Lakewood, NJ, USA, 0.6 mg/mL). Hepatocytes were liberated from liver, washed and seeded in plates with Williams E medium (Sigma-Aldrich, Shanghai, China) supplemented with 6% fetal bovine serum (Lonsa, Richmond, VA, USA). The medium was changed after 2 h and cells were cultured for another 16 h.
Fluorescence MitoTracker Red staining
Mitochondrial staining was performed according to Xue et al. with minor modification[28]. Briefly, hepatocytes were incubated with serum free William's medium E containing 20 nM MitoTracker (Cat. #C1035, Beyotimem, Shanghai, China) in an incubator at 37°C for 15 min. Cells were washed with PBS for three times, and then incubated with 10 μg/ml Hoechst in serum-free William's medium E for 5 min. Following another PBS wash, high-magnification images of the mitochondrial morphology were captured at 63×magnification with the Carl Zeiss LSM710 laser scanning confocal microscope.
Statistical analysis
Data were presented as mean ± standard error of the mean. Comparison between two groups of data was conducted using a two-tailed Student's t-test. One-way analysis of variance was used to compare data across more than two groups. P <0.05 signifies statistical significance in the findings.
Results
SIRT6 deficiency in the liver exacerbated cholestatic liver disease
To investigate the role of SIRT6 in liver injury caused by cholestasis, we used mice with hepatocyte-specific deletion of Sirt6 (Sirt6Δhep) and simulated extrahepatic cholestasis using BDL surgery[25]. It indicated that Sirt6Δhep mice began to exhibit mortality 9 days after BDL. By the 22nd day, their survival rate had fallen to 50%, in contrast to the control mice (Sirt6f/f), which showed no mortality during the same period (Fig. 1A). Hence, we examined liver damage in both groups on the sixth day post-surgery, before the occurrence of death in either mouse group. As shown in Fig. 1B and 1C, compared with the Sirt6f/f group, Sirt6Δhep mice had a significant increase in serum ALT and ALP activities, which are indicators of injury to hepatic parenchymal cells and cholangiocytes. The histological assessment indicated a significant expansion of the necrotic area in the livers of Sirt6Δhep mice, compared with Sirt6f/f mice (Fig. 1D and 1E). As oxidative stress and mitochondrial dysfunction are key pathological mechanisms in cholestatic liver disease[3,29–30], we examined hepatic MDA and ROS levels to assess the degree of oxidative stress, and tested ATP levels to evaluate mitochondrial respiratory chain function. We also paid attention to mtDNA integrity since it houses the genetic information for respiratory chain proteins[3,30]. As shown in Fig. 1F–1I, Sirt6Δhep mice displayed significantly elevated levels of MDA and ROS in their livers, compared with Sirt6f/f mice, accompanied by notable declines in mtDNA copy numbers and ATP levels. Cyt C consists of proteins from the mitochondrial respiratory chain. When the mitochondrial structure is compromised, Cyt C is discharged from the mitochondria into the cytosol, activating the apoptotic pathway downstream. Hence, it plays a pivotal role as an indicator of both mitochondrial structural integrity and apoptosis[31]. After isolating cytoplasm from mouse livers, we performed ELISA to assess the cytoplasmic Cyt C levels, and found that Sirt6Δhep mice following surgery demonstrated higher concentrations of Cyt C in the liver cytoplasm, compared with Sirt6f/f mice (Fig. 1J). The intracellular mitochondrial number, representing the combined result of mitochondrial damage and biogenesis, was detected by fluorescence MitoTracker Red staining. The mitochondrial number of hepatocytes was found to be significantly decreased after BDL. Sirt6 knockdown itself did not affect the mitochondrial number, but in the presence of BDL, the mitochondrial number of Sirt6 knockdown hepatocytes was further decreased, compared with those of the wild ones (Fig. 1K and 1L). Immunoblotting indicated a significant elevation in the hepatic levels of cleaved CASPASE-3 and BAX after BDL, crucial proteins linked to apoptosis. Moreover, the deletion of Sirt6 resulted in a further augmentation of their expression levels (Fig. 1M and 1N). The Sirt6f/f and Sirt6Δhep mice that underwent sham surgery didn't show any significant variations in liver injury, oxidative stress, mitochondrial dysfunction, or apoptosis (Fig. 1B–1N). It appeared that SIRT6 deficiency alone did not set off liver lesions. However, in the presence of cholestasis, Sirt6 deletion intensified liver injury, oxidative stress, mitochondrial loss and dysfunction, and the apoptosis of hepatocytes.
A: Survival curves of Sirt6f/f (n = 10) and Sirt6Δhep (n = 10) mice during the 22 days after BDL. B–J: Sirt6f/f and Sirt6Δhep mice underwent Sham or BDL and were maintained for 6 days. Mice were divided into 4 groups: Sirt6f/f mice + Sham, Sirt6Δhep + Sham, Sirt6f/f + BDL, and Sirt6Δhep+ BDL (n = 7 in each group). B: Serum was harvested for ALT activity assay. C: Serum was harvested for ALP activity assay. D: Livers were fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining. Scale bar, 100 μm. E: Ratio of necrotic area to total liver section area was calculated by Image J. F: Livers were homogenized to assay the MDA levels. G: Livers were homogenized to assay the ROS levels. ROS levels in each group were normalized to the group Sirt6f/f mice + Sham. H: mtDNA was extracted from livers for quantitative PCR. Cox1 was determined as a mtDNA marker with 36B4 as internal standard. I: Livers were homogenized to assay the ATP levels. ATP levels in each group were normalized to the group Sirt6f/f mice + Sham. J: Liver cytosol was extracted for assaying cytosolic Cyt C levels. Cytosolic Cyt C levels in each group were normalized to the group Sirt6f/f mice + Sham. K–N: Sirt6f/f mice + Sham, Sirt6Δhep + Sham, Sirt6f/f + BDL, and Sirt6Δhep+ BDL (n = 7 in each group) were maintained for six days after surgery. K: Hepatocytes isolated from mice underwent fluorescence MitoTracker Red staining. Scale bar, 10 μm. L: The number of mitochondria was quantified from three different hepatocytes. M: The protein levels of SIRT6, cleaved CASPASE-3, and BAX in the liver were determined by immunoblotting. TUBULIN served as the internal control. N: The quantification of bands of SIRT6, cleaved CASPASE-3, and BAX. *P < 0.05. Abbreviations: BDL, bile duct ligation; ALT, aminotransferase; ALP, alkaline phosphatase; MDA, malonaldehyde; Cox1, cytochrome C oxidase 1; Cyt C, cytochrome C.
Hepatic overexpression of SIRT6 alleviated cholestatic liver disease
To further illustrate how SIRT6 functions in combating cholestatic liver disease, we used adenoviral vectors to increase the expression of SIRT6 in mouse liver, and subsequently carried out BDL. The results showed that hepatic overexpression of SIRT6 significantly reversed the BDL-induced effects, including the elevation in serum ALT and ALP levels (Fig. 2A and 2B), the expansion of hepatic necrosis (Fig. 2C and 2D), the increase in liver MDA and ROS levels (Fig. 2E and 2F), and the reduction in mtDNA copy number and hepatic ATP levels (Fig. 2G and 2H). Additionally, it prevented Cyt C release from mitochondria to the hepatocyte cytoplasm (Fig. 2I), reversed BDL-induced downregulation of mitochondrial number (Fig. 2J and 2K), and reduced the expression of cleaved CASPASE-3 and BAX proteins (Fig. 2L and 2M). In the conditions where a sham operation was performed, SIRT6 overexpression in the liver did not show a significant influence on the above-mentioned aspects, and it only slightly decreased the expression of cleaved CASPASE-3 and BAX proteins (Fig. 2L and 2M). These results suggest that hepatic SIRT6 overexpression may protect against liver injury induced by cholestasis, along with preventing oxidative stress, mitochondrial dysfunction, and apoptosis.
Figure
2.
Hepatic overexpression of SIRT6 alleviated cholestatic liver disease in mice.
C57BL/6 mice, infected by adenoviral vector expressing SIRT6 (Ad-Sirt6) or control vector (Ad-Control), after 7 days, underwent Sham or BDL and were maintained for 6 days. Mice were divided into 4 groups: Ad-Control + Sham, Ad-Sirt6 +Sham, Ad-Control + BDL, Ad-Sirt6 + BDL (n = 7 in each group). A: Serum was harvested for ALT activity assay. B: Serum was harvested for ALP activity assay. C: Livers were fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining. Scale bar, 100 μm. D: Ratio of necrotic area to total liver section area was calculated by Image J. E: Livers were homogenized to assay the MDA levels. F: Livers were homogenized to assay the ROS levels. ROS levels in each group were normalized to the group Ad-Control + Sham. G: mtDNA was extracted from livers for quantitative PCR. Cox1 was determined as a mtDNA marker with 36B4 as internal standard. H: Livers were homogenized to assay the ATP levels. ATP levels in each group were normalized to the group Ad-Control + Sham. I: Liver cytosol was extracted for assaying cytosolic Cyt C levels. Cytosolic Cyt C levels in each group were normalized to the group Ad-Control + Sham. J–M: Ad-Control + Sham, Ad-Sirt6 +Sham, Ad-Control + BDL, Ad-Sirt6 + BDL (n = 3 in each group) were maintained for six days after surgery. J: Hepatocytes isolated from mice underwent fluorescence MitoTracker Red staining. Scale bar, 10 μm. K: The number of mitochondria was quantified from three different hepatocytes. L: The protein levels of SIRT6, cleaved CASPASE-3, and BAX in the liver were determined by immunoblotting. TUBULIN served as the internal control. M: The quantification of bands of SIRT6, cleaved CASPASE-3, and BAX. *P < 0.05. Abbreviations: BDL, bile duct ligation; ALT, aminotransferase; ALP, alkaline phosphatase; MDA, malonaldehyde; Cox1, cytochrome C oxidase 1; Cyt C, cytochrome C.
MDL801 activated hepatic SIRT6 to relieve cholestatic liver disease
To assess the therapeutic potential of SIRT6 as a target for cholestasis, we used the SIRT6 agonist MDL801 to treat Sirt6f/f and Sirt6Δhep mice with BDL. Consistant to Fig. 1, Sirt6Δhep mice suffered more severe liver damage than Sirt6f/f mice (Fig. 3). Similar to SIRT6 overexpression, MDL801 effectively counteracted the elevation of serum ALT and ALP levels (Fig. 3A and 3B), reduced the extent of hepatic necrosis (Fig. 3C and 3D), and inhibited the increase in liver MDA and ROS levels (Fig. 3E and 3F) in Sirt6f/f mice. It also increased mtDNA copy number and ATP levels (Fig. 3G and 3H), prevented the leakage of Cyt C into the hepatic cytoplasm (Fig. 3I), reversed the BDL-induced downregulation of mitochondrial number (Fig. 3J and 3K), and decreased the expression of cleaved CASPASE-3 and BAX proteins (Fig. 3L and 3M). The selective activation of SIRT6 by MDL801 were evidecnced through the fact that MDL801 induced a significant decrease in the acetylation levels of histone H3, a substrate for SIRT6, in the livers of Sirt6f/f mice, but this effect was not observed in Sirt6Δhep mice (Fig. 3L and 3M). Notably, MDL801 did not exhibit a significant therapeutic response in bile duct-ligated Sirt6Δhep mice (Fig. 3), implying that its therapeutic efficacy may primarily arise from its targeting of hepatic SIRT6, rather than activation of SIRT6 in other tissues or from non-SIRT6-agonistic-effects.
Figure
3.
MDL801 relieved cholestatic liver disease in a SIRT6-dependent manner.
Sirt6f/f and Sirt6Δhep mice underwent BDL and were maintained for 6 days. Mice, injected intraperitoneally with vehicle or MDL801 (100 mg/kg) 1 day before BDL and daily after BDL, were randomly divided into 4 groups: Sirt6f/f +Vehicle, Sirt6f/f +MDL-801, Sirt6Δhep + Vehicle, Sirt6Δhep + MDL-801 (n = 7 in each group). A: Serum was harvested for ALT activity assay. B: Serum was harvested for ALP activity assay. C: Livers were fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining. Scale bar, 100 μm. D: Ratio of necrotic area to total liver section area was calculated by Image J. E: Livers were homogenized to assay the MDA levels. F: Livers were homogenized to assay the ROS levels. ROS levels in each group were normalized to the group Sirt6f/f +Vehicle. G: mtDNA was extracted from livers for quantitative PCR. Cox1 was determined as a mtDNA marker with 36B4 as internal standard. H: Livers were homogenized to assay the ATP levels. ATP levels in each group were normalized to the group Sirt6f/f +Vehicle. I: Liver cytosol was extracted for assaying cytosolic Cyt C levels. Cytosolic Cyt C levels in each group were normalized to the group Sirt6f/f +Vehicle. J–M: Sirt6f/f +Vehicle, Sirt6f/f +MDL-801, Sirt6Δhep + Vehicle, Sirt6Δhep + MDL-801 were maintained for six days after BDL. J: Hepatocytes isolated from mice underwent fluorescence MitoTracker Red staining. Scale bar, 10 μm. K: The number of mitochondria was quantified from three different hepatocytes. L: The protein levels of SIRT6, Ac-H3K9, H3, cleaved CASPASE-3, and BAX in the liver were determined by immunoblotting. TUBULIN served as the internal control. M: The quantification of bands of SIRT6, Ac-H3K9, H3, cleaved CASPASE-3, and BAX. *P < 0.05. Abbreviations: BDL, bile duct ligation; ALT, aminotransferase; ALP, alkaline phosphatase; MDA, malonaldehyde; Cox1, cytochrome C oxidase 1; Cyt C, cytochrome C; n.s., not significant.
Scavenging hepatic oxidative stress did not compensate for the exacerbation of cholestatic liver disease caused by SIRT6 deficiency
To investigate whether Sirt6's ability to combat oxidative stress contributes to cholestasis treatment, we used a potent antioxidant, NAC, to treat hepatic Sirt6-deficient mice with BDL. NAC did not reverse the increase in serum ALT and ALP levels (Fig. 4A and 4B), or reduce the extent of hepatic necrosis (Fig. 4C and 4D), although it did reduce the elevation of MDA and ROS levels in the livers of Sirt6Δhep mice (Fig. 4E and 4F). In addition, NAC failed to increase the copy number of mtDNA and ATP levels (Fig. 4G and 4H), prevent the release of Cyt C into the liver cytoplasm (Fig. 4I) and decrease the protein levels of cleaved CASPASE-3 and BAX (Fig. 4J and 4K).
Figure
4.
NAC alleviated oxidative stress but not liver injury or mitochondrial dysfunction in mice with cholestatic liver disease.
Sirt6f/f and Sirt6Δhep mice underwent BDL and were maintained for 6 days. Mice, treated intraperitoneally with vehicle or NAC (200 mg/kg) 1 day before BDL and daily after BDL, were randomly divided into 3 groups: Sirt6f/f +Vehicle, Sirt6Δhep + Vehicle, Sirt6Δhep + NAC (n = 7 in each group). A: Serum was harvested for ALT activity assay. B: Serum was harvested for ALP activity assay. C: Livers were fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining. Scale bar, 100 μm. D: Ratio of necrotic area to total liver section area was calculated by Image J. E: Livers were homogenized to assay the MDA levels. F: Livers were homogenized to assay the ROS levels. ROS levels in each group were normalized to the group Sirt6f/f +Vehicle. G: mtDNA was extracted from livers for quantitative PCR. Cox1 was determined as a mtDNA marker with 36B4 as internal standard. H: Livers were homogenized to assay the ATP levels. ATP levels in each group were normalized to the group Sirt6f/f +Vehicle. I: Liver cytosol was extracted for assaying cytosolic Cyt C levels. Cytosolic Cyt C levels in each group were normalized to the group Sirt6f/f +Vehicle. J–K: Sirt6f/f +Vehicle, Sirt6Δhep + Vehicle, Sirt6Δhep + NAC (n = 3 in each group) were maintained for six days after BDL. J: The protein levels of SIRT6, cleaved CASPASE-3, and BAX in the liver were determined by immunoblotting. TUBULIN served as the internal control. K: The quantification of bands of SIRT6, cleaved CASPASE-3, and BAX. *P < 0.05. Abbreviations: BDL, bile duct ligation; NAC, N-acetylcysteine; ALT, aminotransferase; ALP, alkaline phosphatase; MDA, malonaldehyde; Cox1, cytochrome C oxidase 1; Cyt C, cytochrome C; n.s., not significant.
To avoid the potential insufficiency of NAC to eliminate oxidative stress due to the challenge of maintaining consistent blood concentrations through intermittent administration, we used the NRF2 agonist KNI-1 in Sirt6Δhep mice. This treatment led to an increase in the levels of Ho1 and Nqo1, the antioxidant genes as the transcriptional targets of NRF2 in the liver, providing a sustained ability to counteract oxidative stress (Fig. 5A). Just like NAC, KNI-1 did not decrease the serum ALT and ALP levels (Fig. 5B and 5C), reduce the extent of liver necrosis (Fig. 5D and 5E), although it prevented the increase in MDA and ROS levels and partially restored the copy number of mtDNA in the liver of Sirt6Δhep mice (Fig. 5F–5H). In addition , KNI-1 failed to restore ATP levels (Fig. 5I), inhibit the release of Cyt C into the cytoplasm (Fig. 5J), and affect the elevated levels of cleaved CASPASE-3 and BAX proteins (Fig. 5K and 5L). Based on these findings, it appears that attempting to scavenge or inhibit oxidative stress is not sufficient to counteract the exacerbation of cholestatic liver disease resulting from SIRT6 deficiency.
Figure
5.
KNI-1 alleviated oxidative stress and promoted mitochondrial biogenesis but did not relieve liver injury or mitochondrial dysfunction in mice with cholestatic liver disease.
Sirt6f/f and Sirt6Δhep mice underwent BDL and were maintained for 6 days. Mice, treated intraperitoneally with vehicle or KNI-1 (40 mg/kg) 1 day before BDL and daily after BDL, were randomly divided into 3 groups: Sirt6f/f +Vehicle, Sirt6Δhep + Vehicle, Sirt6Δhep + KNI-1 (n = 7 in each group). A: The mRNA levels of Tfam, Ho-1, and Nqo1 were analyzed by RT-qPCR. B: Serum was harvested for ALT activity assay. C: Serum was harvested for ALP activity assay. D: Livers were fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining. Scale bar, 100 μm. E: Ratio of necrotic area to total liver section area was calculated by Image J. F: Livers were homogenized to assay the MDA levels. G: Livers were homogenized to assay the ROS levels. ROS levels in each group were normalized to the group Sirt6f/f +Vehicle. H: mtDNA was extracted from livers for quantitative PCR. Cox1 was determined as a mtDNA marker with 36B4 as internal standard. I: Livers were homogenized to assay the ATP levels. ATP levels in each group were normalized to the group Sirt6f/f +Vehicle. J: Liver cytosol was extracted for assaying cytosolic Cyt C levels. Cytosolic Cyt C levels in each group were normalized to the group Sirt6f/f +Vehicle. K–L: Sirt6f/f +Vehicle, Sirt6Δhep + Vehicle, Sirt6Δhep + KNI-1 (n = 3 in each group) were maintained for six days after BDL. K: The protein levels of SIRT6, cleaved CASPASE-3, and BAX in the liver were determined by immunoblotting. TUBULIN served as the internal control. L: The quantification of bands of SIRT6, cleaved CASPASE-3, and BAX. *P < 0.05. Abbreviations: BDL, bile duct ligation; ALT, aminotransferase; ALP, alkaline phosphatase; MDA, malonaldehyde; Cox1, cytochrome C oxidase 1; Cyt C, cytochrome C; n.s., not significant.
Stimulation of mitochondrial biogenesis did not compensate for the exacerbation of cholestatic liver disease caused by SIRT6 deficiency
The intracellular accumulation of bile acids may disrupt the structure and function of mitochondria, resulting in apoptosis and necrosis. Therefore, restoring mitochondrial structure by promoting mitochondrial biogenesis may potentially offer an effective approach to reverse cholestatic liver disease. It has been reported that SIRT6 enhances the expression of genes involved in mitochondrial biosynthesis, such as Tfam, through the AMPK/PGC1α pathway. This mechanism helps defend biliary endothelial cells against bile acid-induced apoptosis[17]. Consistent with this finding, we found that hepatic overexpression of SIRT6 triggered the AMPK/PGC1α pathway and increased the expression levels of genes related to mitochondrial biosynthesis, including Tfam and Idh2 (Fig. 6A and 6B). To investigate the role of mitochondrial biogenesis in SIRT6's resistance against cholestatic liver disease, we administered the AMPK agonist AICAR to Sirt6Δhep mice, and observed a significant restoration of the AMPK phosphorylation and PGC1α protein levels that were impaired by Sirt6 deletion. This intervention also resulted in increased transcript levels of Tfam and Idh2 (Fig. 6C and 6D). Despite this, AICAR did not significantly improve serum ALT and ALP levels, hepatic necrosis area, MDA and ROS levels, mtDNA copy number, ATP levels, hepatocyte cytoplasmic Cyt C levels, or cleaved CASPASE-3 and BAX protein levels (Fig. 6E–6O). The results indicate that enhancing mitochondrial biosynthesis through the AMPK/PGC1α pathway is not a critical requirement for SIRT6 to exert its anticholestatic function.
Figure
6.
AICAR failed to alleviate cholestatic liver injury, oxidative stress, and mitochondrial dysfunction in mice.
A–B: C57BL/6 mice, infected by adenoviral vector expressing SIRT6 (Ad-Sirt6) or control vector (Ad-Control), after 7 days, underwent Sham or BDL and were maintained for 6 days. All animals were divided into 4 groups: Ad-Control + Sham, Ad-Sirt6 +Sham, Ad-Control + BDL, Ad-Sirt6 + BDL (n = 7 in each group). A: Immunoblotting analysis of p-AMPK, AMPK, and PGC1α protein expression in liver. B: The mRNA levels of Tfam and Idh2 were analyzed by RT-qPCR. C-M: Sirt6f/f and Sirt6Δhep mice underwent BDL and were maintained for 6 days. Mice, treated intraperitoneally with vehicle or AICAR (250 mg/kg) 1 day before BDL and daily after BDL, were randomly divided into 3 groups: Sirt6f/f +Vehicle, Sirt6Δhep + Vehicle, Sirt6Δhep + AICAR (n = 7 in each group). C: Immunoblotting analysis of p-AMPK, AMPK, and PGC1α protein expression in the liver. D: The mRNA levels of Tfam and Idh2 were analyzed by qPCR. E: Serum was harvested for ALT activity assay. F: Serum was harvested for ALP activity assay. G: Livers were fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining. Scale bar, 100 μm. H: Ratio of necrotic area to total liver section area was calculated by Image J. I: Livers were homogenized to assay the MDA levels. J: Livers were homogenized to assay the ROS levels. ROS levels in each group were normalized to the group Sirt6f/f +Vehicle. K: mtDNA was extracted from livers for quantitative PCR. Cox1 was determined as a mtDNA marker with 36B4 as internal standard. L: Livers were homogenized to assay the ATP levels. ATP levels in each group were normalized to the group Sirt6f/f +Vehicle. M: Liver cytosol was extracted for assaying cytosolic Cyt C levels. Cytosolic Cyt C levels in each group were normalized to the group Sirt6f/f +Vehicle. N-O: Sirt6f/f +Vehicle, Sirt6Δhep + Vehicle, Sirt6Δhep + AICAR (n = 3 in each group). N: The protein levels of SIRT6, cleaved CASPASE-3, BAX in the liver were evaluated by immunoblotting. TUBULIN served as the internal control. O: The quantification of bands of SIRT6, cleaved CASPASE-3, and BAX. *P < 0.05. Abbreviations: BDL, bile duct ligation; ALT, aminotransferase; ALP, alkaline phosphatase; MDA, malonaldehyde; Cox1, cytochrome C oxidase 1; Cyt C, cytochrome C, n.s., not significant.
SIRT6 primarily reduced bile acid synthesis to alleviate cholestatic liver disease
Reducing bile acid production is a successful approach to ameliorating liver damage caused by cholestasis. The serum level of bile acid may be elevated because of the release of bile acids into the bloodstream as a result of impaired enterohepatic circulation or overproduction of bile acids. In the mice with BDL, Sirt6 deficiency in the liver led to a notable increase in the levels of serum bile acids and hepatic CYP7A1, a crucial enzyme responsible for synthesizing bile acids (Fig. 7A). While, elevated expression of hepatic SIRT6 or MDL801, a SIRT6 agonist, significantly reduced the CYP7A1 expression and serum bile acid levels (Fig. 7B and 6C). This suggests that SIRT6 reduces bile acid synthesis by inhibiting the expression of CYP7A1, thereby alleviating cholestasis. We also tested the effect of KNI-1, AICAR, and NAC, those substances affecting mitochondrial biosynthesis or oxidative stress. As a result, none of them inhibited the expression of CYP7A1 or reduced the concentration of bile acids in the bloodstream (Fig. 7D–7F).
Figure
7.
MDL801 rather than KNI-1, AICAR, or NAC reduced bile acid synthesis in mice with cholestatic liver disease.
A: Sirt6f/f and Sirt6Δhep mice underwent BDL and were maintained for 6 days (n = 6 in each group). B: C57BL/6 mice, infected by adenoviral vector expressing SIRT6 (Ad-Sirt6) or control vector (Ad-Control), after 7 days, underwent BDL and were maintained for 6 days (n = 6 in each group). C: Sirt6f/f mice underwent BDL and were maintained for 6 days. Mice, treated intraperitoneally with vehicle or MDL801 (100 mg/kg) 1 day before BDL and daily after BDL, were randomly divided into 2 groups: Sirt6f/f +Vehicle, Sirt6f/f +MDL-801 (n = 6 in each group). D: Sirt6f/f mice underwent BDL and were maintained for 6 days. Mice, treated intraperitoneally with vehicle or NAC (200 mg/kg) 1 day before BDL and daily after BDL, were randomly divided into 2 groups: Sirt6f/f +Vehicle, Sirt6f/f + NAC (n = 6 in each group). E: Sirt6f/f mice underwent BDL and were maintained for 6 days. Mice, treated intraperitoneally with vehicle or KNI-1 (40 mg/kg) 1 day before BDL and daily after BDL, were randomly divided into 2 groups: Sirt6f/f +Vehicle, Sirt6f/f + KNI-1 (n = 7 in each group). F: Sirt6f/f mice underwent BDL and were maintained for 6 days. Mice, treated intraperitoneally with vehicle or AICAR (250 mg/kg) 1 day before BDL and daily after BDL, were randomly divided into 2 groups: Sirt6f/f +Vehicle, Sirt6f/f + AICAR (n = 6 in each group). The protein levels of CYP7A1 in the liver were determined by immunoblotting. TUBULIN served as the internal control. Serum was harvested for bile acid level assay. *P < 0.05. Abbreviations: BDL, bile duct ligation; NAC, N-acetylcysteine; KNI-1, Keap1-Nrf2-IN-1; AICAR, acadesine; n.s., not significant.
To further validate the importance of down-regulating CYP7A1 in the anticholestatic effects of SIRT6. We used adeno-associated viral vectors to specifically knock down CYP7A1 in the livers of Sirt6f/f and Sirt6Δhep mice with BDL. Consistant to Fig. 1, Sirt6Δhep mice got severer cholestasis than Sirt6f/f mice, when receiving shControl (Fig. 8). While hepatic CYP7A1 knockdown significantly relieved BDL-induced cholestasis in Sirt6f/f mice. Sirt6f/f mice receving shCyp7A1 had lower serum ALT and ALP levels (Fig. 8A and 8B), less hepatic necrosis (Fig. 8C and 8D), lower liver MDA and ROS levels (Fig. 8E and 8F), more mtDNA copy number (Fig. 8G), higher ATP level (Fig. 8H), less Cyt C entering the hepatocyte cytoplasm (Fig. 8I), more mitochondrial number in hepatocytes (Fig. 8J and 8K), and lower levels of cleaved CASPASE-3 and BAX proteins (Fig. 8L and 8M). Furthermore, hepatic CYP7A1 knockdown smoothed out the differences between Sirt6f/f and Sirt6Δhep mice based on these metrics (Fig. 8).
Figure
8.
Hepatic knockdown of CYP7A1 counteracted cholestatic liver injury caused by hepatic BDL and Sirt6 deficiency.
Sirt6f/f or Sirt6Δhep mice, infected either by adeno-associated virus knocking down Cyp7A1 (shCyp7A1) or control vector (shControl), after three weeks, underwent BDL and were maintained for six days. Mice were divided into 4 groups: Sirt6f/f + shControl, Sirt6Δhep + shControl, Sirt6f/f + shCyp7A1, Sirt6Δhep + shCyp7A1 (n = 7 in each group). A: Serum was harvested for ALT activity assay. B: Serum was harvested for ALP activity assay. C: Livers were fixed in 4% paraformaldehyde and embedded in paraffin for H&E staining. Scale bar, 100 μm. D: Ratio of necrotic area to total liver section area was calculated by Image J. E: Livers were homogenized to assay the MDA levels. F: Livers were homogenized to assay the ROS levels. ROS levels in each group were normalized to the group Sirt6f/f + shControl. G: mtDNA was extracted from livers for quantitative PCR. Cox1 was determined as a mtDNA marker with 36B4 as internal standard. H: Livers were homogenized to assay the ATP levels. ATP levels in each group were normalized to the group Sirt6f/f + shControl. I: Liver cytosol was extracted for assaying cytosolic Cyt C levels. Cytosolic Cyt C levels in each group were normalized to the group Sirt6f/f + shControl. J–M: Sirt6f/f + shControl, Sirt6Δhep + shControl, Sirt6f/f + shCyp7A1, Sirt6Δhep + shCyp7A1 (n = 3 in each group) were maintained for six days after BDL. J: Hepatocytes isolated from mice underwent fluorescence MitoTracker Red staining. Scale bar, 10 μm. K: The number of mitochondria was quantified from three different hepatocytes. L: The protein levels of SIRT6, cleaved CASPASE-3, and BAX in the liver were determined by immunoblotting. TUBULIN served as the internal control. M: The quantification of bands of SIRT6, cleaved CASPASE-3, and BAX. *P < 0.05. Abbreviations: BDL, bile duct ligation; ALT, aminotransferase; ALP, alkaline phosphatase; MDA, malonaldehyde; Cox1, cytochrome C oxidase 1; Cyt C, cytochrome C; n.s., not significant.
The present study demonstrated that SIRT6 acts as a protective factor against BDL-induced cholestatic liver disease. Based on the existing research and experimental findings, we proposed that SIRT6 might potentially deliver its therapeutic effects by reducing oxidative stress, promoting mitochondrial biogenesis, or suppressing the production of bile acids. By analyzing the three pathways, we discovered that the blocking of bile acid production might be the primary mechanism underlying the anticholestatic properties of SIRT6.
Bile acids interfere with the functioning of large conductance channels located in the mitochondrial inner membrane, specifically those sensitive to cyclosporine A/trifluoperazine. This disruption triggers the opening of the mitochondrial permeability transition pore, resulting in the collapse of the mitochondrial inner transmembrane potential, expansion of the matrix space, and eventual rupture of the outer membrane. The collapse of the mitochondrial transmembrane potential disconnects the respiratory chain, preventing the production of ATP and promoting the generation of ROS. When the mitochondrial membrane breaks, it results in the release of mitochondrial proteins like Cyt C. Furthermore, bile acids induce conformational alterations in a pro-apoptotic protein, BAX, prompting its shift from the cytoplasm to the mitochondria, and thus initiating the release of Cyt C. When Cyt C is released, cell death may occur either through an apoptotic mechanism involving caspase activation or through necrosis arising from the disruption of electron transport[31].
When ROS levels increase, cells experience oxidative stress, leading to damage to DNA, proteins, and lipids, and ultimately affecting the integrity of cell membranes and inducing apoptosis[30]. However, our experiments showed that treating cholestasis-induced liver injury with anti-oxidative stress methods alone did not lead to a reversal of this condition. It is speculated that oxidative stress, as a downstream event of compromised mitochondrial membrane potential and structural anisotropy, even though diminished, cannot halt the upstream mitochondrial damage or mitochondrial damage-triggered apoptosis and necrosis.
Mitochondrial biogenesis of hepatocytes was severely impaired in cholestatic liver disease. PGC1α, the key transcriptional activator in mitochondrial biogenesis[19], acts to increase the production of mitochondrial components such as IDH2, cytochrome C oxidase subunit Ⅳ (COX Ⅳ), and Cyt C. Additionally, PGC1α enhances the expression of the transcription factor NRF2, triggering the transcription of Tfam[17,32]. TFAM plays a pivotal role by binding to mtDNA, contributing to its stabilization, replication, and transcription of mtDNA[33–34]. It's worth noting that mtDNA encodes crucial subunits of the respiratory chain complex, which plays a pivotal role in the process of mitochondrial biogenesis[4–5]. When bile acids accumulate, they block the expression of PGC1α and its downstream genes, including Nrf2, Tfam, and various mitochondrial proteins[35]. Therefore, increasing levels of SIRT6 and using a SIRT6 agonist might reverse this inhibition of those mitochondrial biogenesis-related genes. To investigate whether mitochondrial biogenesis affects SIRT6's resistance to cholestatic liver disease, we administered AICAR to liver-specific Sirt6 knockout mice to activate the AMPK/PGC1α pathway, or used KNI-1 to activate NRF2. Although these approaches increased the expression levels of genes related to mitochondrial biogenesis, they didn't counteract the exacerbation of liver damage following the Sirt6 knockdown. It is evident that mitochondrial biosynthesis may not be the main pathway through which SIRT6 mitigates cholestatic liver disease. The accumulation of bile acids in liver cells likely causes continuous damage to newly developed mitochondria, so increasing mitochondrial production does not improve overall mitochondrial function.
It has been reported that SIRT6 inhibits Cyp7A1 transcription, thereby decreasing bile acid synthesis and thereby alleviating liver injury associated with cholestasis[20]. Considering neither oxidative stress nor mitochondrial biogenesis serves as a pathway for SIRT6 in combating cholestatic liver disease, we theorized that the inhibition of bile acid synthesis might be the key mechanism underlying the SIRT6's therapeutic function. Supporting this, the SIRT6 agonist, MDL801, as opposed to NAC, KNI-1, or AICAR, reduced the expression level of hepatic CYP7A1 and the concentration of bile acids in the serum in BDL mice. In addition, the knockdown of hepatic CYP7A1, which serves as a key enzyme in the classical synthesis pathway for bile acids[36], greatly alleviated BDL-induced cholestasis and counteracted the adverse effects of Sirt6 deletion.
Impeding the synthesis of bile acids by down-regulation of CYP7A1 has become a widely adopted approach in the treatment of cholestatic liver diseases. Taking obeticholic acid, cilofexor, and tropifexor as examples, these steroidal and non-steroidal farnesoid X receptor (FXR) agonists activate the FXR/small heterodimer partner (SHP) pathway and thereby inhibit hepatocyte nuclear factor 4α (HNF4α)-mediated transcription of Cyp7A1[37–39]. Similarly, alafermin, a fibroblast growth factor 19 (FGF19) analog, disrupts transcription factor EB (TFEB)-mediated transcription of Cyp7A1 through the mechanistic target of rapamycin/extracellular signal-regulated kinase pathway[37,40]. Fibrates stimulate peroxisome proliferator-activated receptor (PPARα), resulting in the transcriptional repression of Cyp7A1[37,41]. Activation of SIRT6 reduces Cyp7A1's transcription by inhibiting estrogen-related receptors (ERRγ)[20], which is a completely novel regulatory pathway. Therefore, SIRT6 agonists may be highly synergistic with the above drugs, allowing for lower drug dosages, thereby reducing adverse effects, such as dyslipidemia and itchiness by FXR agonists, carcinogenic risks by FGF19 analogs, and myalgia as well as rhabdomyolysis by PPARα agonists[37].
The intestinal flora is also deeply involved in the bile acid metabolism. Gut microbiota with bile salt hydrolase activity could remove the glycine or taurine from conjugated bile acids to increase their hydrophobicity, thereby facilitating the fecal elimination of bile acids[42]. This function is synergistic with SIRT6 agonists potentially beneficial for resistance to bile toxicity and cholestasis. Notably, bile acids play a role in shaping the gut microbiome by promoting the growth of bile-acid-metabolizing bacteria and inhibiting the development of other bacteria that are sensitive to bile[42]. Thus, cholestasis or SIRT6 agonists, which likely interfere with the flow of bile acids into the small intestine, may reduce the population of bile acid-metabolizing bacteria population but lead to an overgrowth of other harmful bacteria in the gut. Therefore, during the administration of SIRT6 agonists for cholestasis treatment, it is beneficial to moderately increase the population of bile-acid-metabolizing bacteria and maintain intestinal flora balance, as this may increase the efficacy of the agonists and lessen potential negative effects.
Fundings
This work is supported by the National Natural Science Foundation of China (Grant. No. 82170877).
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