| Objective: To investigate the fluctuation of bile acids in enterohepatic circulation of rats with hemorrhagic shock,and to further investigate whether bile acid could attenuate hemorrhagic shock induced liver injury by activation of SIRT1-FXR pathway to regulate inflammation,apoptosis and proliferation.To investigate the changes of bile acid concentration in bile,serum and liver of Sprague Dawley(SD)rats after hemorrhagic shock,and to screen out specific types of bile acid with potential protective effect.Then explore whether this type of bile acid can protect the liver function of hemorrhagic shock rats with in vitro and in vivo studies.Finally,to elucidate that TUDCA protects liver function by activating sirtuin1(SIRT1)-farnesoid X receptor(FXR)pathway to regulate inflammation,apoptosis and proliferation of hepatocytes.Methods: Rat hemorrhagic shock model was established by femoral artery blood withdraw.The bile,serum and liver were collected at 6,12,and 24 h after hemorrhagic shock.Utra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was applied for bile acid profiling.The specific types of bile acids with potential protective effects were screened out by the change trend of bile acids combined with the analysis of literature reports.Human liver tumor cell line Hep G2 cells were cultured in vitro under hypoxic conditions and stimulated by SIRT1 agonist SRT1720 or tauroursodeoxycholic acid(TUDCA,the specific type of bile acid).After culture,the cell protein and m RNA were extracted and the supernatant was collected.The protein expression of SIRT1,FXR,nuclear factor-? B(NF-? B),tumor protein p53(p53),forkhead box protein M1(Fox M1)in Hep G2 cells were detected by Western Blot.The expression of m RNA in SIRT1 and FXR was detected by reverse transcriptase polymerase chain reaction(RT-PCR).Alanine transaminase(ALT),aspartate transaminase(AST),tumor necrosis factor-alpha(TNF-?),interleukine 6(IL-6),interleukine 1 beta(IL-1?),interleukine 10(IL-10)in cell culture supernatant were analyzd by enzyme-linked immunosorbent assay ELISA).To investigate the protective effect of TUDCA on hypoxic cultured hepatocytes and its potential mechanism.FXR inhibitor Z-guggulsterone was used to inhibit the expression of FXR,and then the expression of SIRT1,FXR and downstream genes in Hep G2 cultured under hypoxic condition was detected after TUDCA intervention.Rat hemorrhagic shock model of was established.One group of rats was pretreated with EX527 to inhibit SIRT1,and TUDCA intervention(50 mg/kg)or SRT1720(30 mg/Kg)was administrated during resuscitation.All rats were sacrificed 24 hours after resuscitation,and the protein expression of SIRT1,FXR,NF-?B and p53 in liver,liver histopathological damage,hepatocytes apoptosis and proliferation,inflammatory cytokines in serum(tumor necrosis factor-?,interleukin-6,interleukin-1?,interleukin-10),and serum biochemical indicators(aspartate aminotransferase,alanine aminotransferase)(n=6)were detected.Results: Compared with control group,bile acids in the bile,serum and liver of rats with hemorrhagic shock did not change significantly at 6 h after shock,but changed significantly at 12 h and 24 h after shock.Among the bile acids in the bile,the taurine conjugated bile acids(T-BAs)decreased,while the proportion of glycine conjugated bile acids(G-BAs)increased.In serum,the dominant unconjugated bile acids(un-BAs)decreased,while G-BAs increased.In the liver,the proportion of un-BAs increased,while T-BAs and G-BAs decreased significantly.In the liver,the change trend of TUDCA decreased significantly with the prolongation of post-shock time.Combined what we found with literature analysis,we speculated that TUDCA was a specific type of bile acid with potential to protect liver in the early stage of hemorrhagic shock.The m RNA and protein levels of SIRT1,FXR,NF-?B and p53 decreased significantly in Hep G2 cells under hypoxic culture,which were consistent with the trend of weakened liver cell proliferation,increased apoptosis and increased inflammatory response(p<0.05).The administration of TUDCA or SIRT1 agonist SRT1720 could significantly activate and up-regulate the expression of SIRT1 and FXR,promoting the expression of Fox M1 and inhibiting the protein expression and acetylation of NF-? B and p53,and thus restored the proliferation activity of liver cells and inhibited hepatocyte apoptosis and inflammatory response(p < 0.05).When Z-guggulsterone was used to inhibit the expression of FXR,the protective effect of TUDCA was weakened(p < 0.05).Hemorrhagic shock reduced the expression of SIRT1 and FXR in rats,while TUDCA and SRT720 could reconstitute SIRT1-FXR signaling,inhibiting NF-?B and p53 expression(p<0.05).TUDCA adminstration restored hepatocyte regeneration,inhibited hepatocyte apoptosis and decreased serum cytokine level,and improved serum biochemical indexes(p < 0.05).When EX527 was used in advance to inhibit SIRT1,the regulatory effect of TUDCA on SIRT-FXR pathway and its downstream signals was significantly weakened,and its effect on improving liver dysfunction in the early stage of hemorrhagic shock was also significantly weakened.Conclusion: The bile acids in the entrohepatic circulation of rats with hemorrhagic shock are significantly changed,in which TUDCA is significantly decreased.Supplementation of TUDCA can protect the liver function in the early stage of hemorrhagic shock.Hypoxia and hemorrhagic shock inhibit the activity of SIRT1-FXR signaling pathway in hepatocytes,which aggravated the inflammatory response and apoptosis of hepatocytes and inhibited hepatocyte regeneration,leading to hypoxia-induced hepatocyte injury and hemorrhagic shock-induced liver dysfunction.TUDCA can activate SIRT1-FXR signal transduction,then promote the expression of Fox M1 protein,inhibit the expression and acetylation of NF-?B and p53 protein,thus promote the proliferation of hepatocytes,inhibit their inflammatory response and apoptosis,and protect the liver function of hemorrhagic shock rats. |
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