| Following injuries by physical,chemical or biological hazards,the remnant normal hepatocytes may compensate for the lost tissue,regain its original mass,and restore the liver function by entering the cell cycle and promoting cell proliferation.This phenomenon is termed as liver regeneration(LR),a complex and dynamic process that involves multiple pathways and factors.LR has been well studied in the rodent model induced by two-thirds partial hepatectomy(PH),which was reported for the first time by Higgins et al.in 1931.In addition,biliary ligation model,chemical-induced hepatotoxic injury or pharmacological model and transgenic animal model are also widely used.Clinically,PH has become a radical treatment for many patients with liver tumor,but the patients with liver tumor are often complicated with hepatitis,liver fibrosis and even cirrhosis caused by different etiologies.Especially in patients with cirrhosis,the ability of LR is significantly reduced,which increases the incidence and mortality of liver failure after PH.Therefore,improving LR ability is the basis for the favourable treatment outcome of patients after PH,which can serve as a potential indicator for postoperative survival.It had been proposed that collagen degradation,liver cell regeneration and blood flow reconstruction are the three major mechanisms of liver fibrosis reversal.Therefore,the reversal of liver fibrosis may depend on the improvement of LR.Traditional Chinese medicine possesses unique advantages in anti-fibrosis.Most of the previous works have emphasized that traditional Chinese medicine such as Salvia miltiorrhiza/Danshensu,Red Peony and Fuzheng Huayu decoction(FZHY)can promote the hepatocyte proliferation and regeneration.Yiqi Huoxue recipe(YQHX)is mainly composed of Salvia miltiorrhiza,red peony root,astragalus root and other Chinese herbs.Our previous work has demonstrated that YQHX may be an effective therapeutic approach for liver fibrosis induced by carbon tetrachloride(CCl4)in rats via regulating hepatocyte autophagy,inhibiting hepatic stellate cell activation and reducing collagen deposition.However,there is still a lack of research on liver regeneration.In this study,we assessed the effect of YQHX on LR in patients with cirrhosis caused by chronic hepatitis B,investigated the potential role of YQHX during LR following two-thirds PH in rats,and further elucidated its underlying mechanism.Furthermore,we assessed the effect of YQHX on LR in the rat model of CCl4-induced cirrhosis.Part One The clinical study of Yiqi Huoxue Recipe in improving liver fibrosis by promoting liver regenerationObjective: To verify the clinical effect of YQHX in promoting LR and improving liver fibrosis.Methods: We retrospectively analyzed 13 patients with liver fibrosis(F?2)caused by chronic hepatitis B who received entecavir(ETV)combined with YQHX(YQHX + ETV group),and 18 patients with chronic hepatitis B who received entecavir alone(ETV group).Hepatic inflammation and fibrosis were observed by hematoxylin and eosin(HE)and Masson's trichromatism staining(Masson)stained liver sections.The expression levels of Cyclin D1,ki67 and proliferating cell nuclear antigen(PCNA)were determined by immunohistochemical staining.The liver biochemistry,liver fibrosis reversal and liver regeneration were compared before and after treatment.Results: After treatment,the YOHX + ETV and ETV groups showed significantl decreases in the serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and gamma glutamyl transpeptidase(P <0.05),and increase in the serum level of albumin(P < 0.05).The fibrosis stage score in the YQHX + ETV group was significantly decreased(P < 0.05),while there was no significant difference in the ETV group(P > 0.05).YQHX and ETV combination administration significantly upregulated the liver expression levels of Cyclin D1,ki67 and PCNA(P < 0.05).Compared with baseline,the ETV group showed significant increase in the liver expression levels of Cyclin D1(P < 0.05),significant decrease in ki67(P < 0.05),while no statistic changes in PCNA(P > 0.05)at end of the treatment.Summary: YQHX combined with ETV can significantly improve LR in patients with liver fibrosis caused by chronic hepatitis B,and significantly improved the liver fibrosis.Part Two Effects of Yiqi Huoxue Recipe on liver regeneration in rats after partial hepatectomyObjective: To determine the effects of YQHX on LR in rats after PH.Methods: Wistar rats were randomly divided into four groups:Sham-operated,PH,YQHX + PH,and FZHY + PH groups(n = 12 in each group).Two-thirds PH was performed according to the method of Higgins and Anderson.The rats in the YQHX + PH and FZHY + PH groups were administered with YQHX(0.50 ml/100 g)and FZHY(0.50 ml/100 g),respectively,by gavage once daily at 3 days prior to PH until the termination of this study.Meanwhile,the rats in the Sham-operated and PH groups were administered once daily with 0.90% sodium chloride solution(0.50 ml/100 g)via oral gavage.All rats were sacrificed under anesthesia at 24 h,72h,96 h and168h after surgery.The rates of LR were calculated,and the expression levels of Cyclin D1 were determined by immunohistochemical staining.Results:1.YQHX significantly increased the liver regeneration rates in rats after PH.After PH operation,the liver regeneration rates of rats in all groups showed an increasing trend with time prolongation.The overall trends of YQHX + PH group and FZHY + PH group were significantly higher than that of PH group,with no significant difference between the two groups(P > 0.05).Compared to PH group(14.20 ± 2.02%),the rates of LR in the YQHX + PH group(25.41 ± 4.03%)and FZHY + PH groups(30.86 ± 0.11%)were markedly increased at 24 h after PH(P < 0.05).At 96 h following PH,the rate of LR in the YQHX + PH group was significantly higher compared to the PH group(56.97 ± 7.69% vs.38.96 ± 4.21%,P < 0.05),while the rate of LR in the FZHY + PH group was significantly higher compared to the PH group(65.80 ± 3.37% vs.53.02 ± 4.32%,P < 0.05)at 168 h after PH.2.YQHX significantly enhanced the expression of Cyclin D1 in the liver of rats after PH.At 24 h,72 h,96 h and 168 h after PH,the immunohistochemical nuclear expression of Cyclin D1 in the PH group was significantly higher than that in the Sham group(P < 0.05).At 24 h after PH,YQHX and FZHY administration increased the expression of Cyclin D1,but no significant difference was found compared with the PH group(P > 0.05).At 72 h and 96 h after PH,YQHX administration slightly increased the positive rate of Cyclin D1 compared to the PH group,but the difference was not statistically significant(P > 0.05).While,the FZHY + PH group showed predominantly increase in the positive rate of Cyclin D1 compared to the PH and YQHX +PH groups(P < 0.05).At 168 h after PH,the expression of Cyclin D1 in liver of rats in all groups was decreased compared with the corresponding previous time node,and the expressions of Cyclin D1 in the YQHX + PH and FZHY +PH groups were increased compared with the Sham group,but there was no statistical difference compared with the PH group(P > 0.05).Summary: YQHX can significantly increase the liver regeneration rate and the expression level of Cyclin D1 in liver tissue of rats after PH surgery,promote the division and proliferation of liver cells,and restore the original volume and function rapidly.Part Three Molecular mechanism of Yiqi Huoxue Recipe in improving liver regeneration in rats after partial hepatectomyObjective: To investigate the mechanism of YQHX during LR following two-thirds PH in rats.Methods: The experimental design was the same as the second part in methods.The expression levels of c-jun were determined by immunohistochemical staining.The protein levels of c-Jun N-terminal kinase(JNK)1/2,p-JNK1/2,c-jun,p-c-jun,B cell lymphoma-2(Bcl-2)and Bcl-2associated x-protein(Bax)were detected by Western blotting,while the m RNA levels of JNK1,JNK2,c-jun,Bcl-2 and Bax were examined by real-time polymerase chain reaction(RT-PCR).Results:1.Immunohistochemical profiles of c-Jun in liver tissueThe immunohistochemical nuclear expression of c-jun was predominantly upregulated in the PH group than that in the Sham group at 24 h,72 h and 96 h after PH(P < 0.05),while no significant difference was found at 168 h between the two groups(P > 0.05).At 24 h after PH,compared with the PH group,the YQHX + PH group exhibited lower expression level of c-jun in rat liver without statistically significance(P > 0.05),while the FZHY+ PH group showed significant lower expression level of c-jun in rat liver(P <0.05).At 72 h after PH,the YQHX + PH group showed significant lower expression level of c-jun in rat liver compared with the PH and FZHY + PH groups(P < 0.05).At 96 h after PH operation,the expressions of c-jun in liver were significantly upregulated in the YQHX + PH and FZHY + PH groups than that in the PH group(P < 0.05),and the expression of c-jun in the YQHX+PH group was significantly higher than that in the FZHY + PH group(P < 0.05).At 168 h after PH surgery,the expressions of c-jun in all groups were decreased,and the FZHY + PH group exhibited higher expression level of c-jun in rat liver compared to the PH and YQHX + PH group(P < 0.05).2.Hepatic m RNA and protein expression levels of JNK signaling pathway-related genesNo significant differences in the hepatic m RNA expression levels of JNK1/2 and c-jun were observed among the four groups.Through the detection of JNK1/2 and c-Jun protein levels,it was found that the phosphorylation index plays a leading role in LR.Compared to the Sham group,the PH group showed markedly increased protein levels of p-JNK1/2 in the liver tissue at 24 h,72 h and 168 h after PH(P < 0.05).Notably,the YQHX + PH and FZHY + PH groups demonstrated significantly upregulated protein expression levels of p-JNK1/2 compared to the PH group(P < 0.05),and its phosphorylation level was much higher in the FZHY + PH group than that in the YQHX + PH group(P < 0.05).At 96 h after PH,the PH group showed markedly increased protein level of p-JNK1/2in the liver tissue compared with the Sham group(P < 0.05),and the FZHY +PH group demonstrated significantly upregulated protein expression levels of p-JNK1/2 compared to the PH and YQHX + PH groups(P < 0.05),while no significant difference was observed between the YQHX + PH and PH goups(P > 0.05).At 72 h after PH,the protein expression level of p-c-jun in the PH group was significantly higher than that in the Sham group(P < 0.05).Compared to the PH group,the protein expression levels of p-c-jun were significantly upregulated in the YQHX + PH and FZHY + PH groups(P < 0.05),and its expression level in the FZHY + PH group was slightly higher than that in the YQHX + PH group without statistically significance(P > 0.05).A similar trend was observed for the expression level of p-c-jun at 24 h after PH,but there was no statistically significant difference among the four groups(P >0.05).At 96 h and 168 h after PH,the protein expression level of p-c-jun in the PH group was significantly higher than that in the Sham group(P < 0.05).The FZHY + PH group showed significant higher level of p-c-jun compared with the PH and YQHX + PH group(P < 0.05),while no significant difference was observed between the YQHX + PH and PH groups(P > 0.05).3.Hepatic m RNA and protein expression levels of apoptosis-related markersThe PH group significantly reduced the protein expression levels of Bax compared to the Sham group at 24 h,72 h,96 h and 168 h after PH(P < 0.05),FZHY administration markedly downregulated the protein expression levels of Bax compared to the PH and YQHX + PH groups(P < 0.05).Notablely,compared with the PH group,the YQHX + PH group showed significantly lower Bax protein expression levels at 24 h and 96 h after PH operation(P <0.05),but no significant difference at 72 h and 168 h after PH operation(P >0.05).The hepatic m RNA expression levels of Bax had the similar trend with its protein expression levels.The PH group significantly induced the protein expression levels of Bcl-2compared to the Sham group at 24 h,72 h,96 h and 168 h after PH(P < 0.05).Notably,the YQHX + PH and FZHY + PH groups showed significant increases in the protein expression levels of Bcl-2 compared to the PH group at 24 h,72 h and 96 h after PH(P < 0.05),while only FZHY administration significantly increased the protein expression level of Bcl-2 when compared with untreated PH group at 168 h after PH(P < 0.05).The protein expression level of Bcl-2 in the YQHX + PH group was significantly higher than that in the FZHY + PH group at 24 h after PH surgery(P < 0.05).Beside that,it was significantly lower than that in the FZHY + PH group at other time points(P <0.05).The m RNA expression trend of Bcl-2 was similar to its protein expression trend.Summary:1.YQHX can significantly increase the protein expression levels of p-JNK1/2 and p-c-jun in rats after PH,and promote liver regeneration via the JNK pathway.2.YQHX recipe significantly inhibited the apoptosis by reducing the expression levels of Bax and upregulating the expression levels of Bcl-2,thus promoting LR in rats after PH.Part Four Effect and mechanism of Yiqi Huoxue Recipe in improving liver fibrosis by promoting liver regenerationObjective: To investigate the effect of YQHX on LR in liver cirrhosis.Methods: Thirty-six male SPF Wistar rats were randomly divided into four groups: i)control group,rats were administered with olive oil(0.20ml/100 g)twice a week by intraperitoneal injection,and 0.90% sodium chloride solution(1 ml/100 g)once daily by gavage;ii)CCl4 group,rats were administrated with a mixture of CCl4(30%)and olive oil(70%)(0.20 ml/100g)twice a week by intraperitoneal injection,and 0.90% sodium chloride solution(1 ml/100 g)once daily by gavage;iii)CCl4 + YQHX group,rats were treated with YQHX(3.40g/10ml/kg·d)by gavage from the beginning of CCl4 injection;?)CCl4 + FZHY group,rats were treated with FZHY(0.46g/10ml/kg·d)by gavage from the beginning of CCl4 injection.All rats were sacrificed at the end of 8 weeks,blood and liver samples were obtained.The serum levels of ALT and AST were tested by enzymic method.Hepatic inflammation and fibrosis of rats were observed by hematoxylin and eosin(HE)and Masson's trichromatism staining(Masson).The expression levels of Cyclin D1 were determined by immunohistochemical staining and western blot.The expression levels of cytokeratin 19(CK19),ki67 and PCNA were determined by immunohistochemical staining.Results:1.YQHX significantly reduced the serum ALT and AST levels in CCl4-induced cirrhosis rat model.Compared with the control group,the CCl4 group showed significant increases in the serum ALT(80.14 ± 35.73 U/L vs.41.43 ± 9.76 U/L)and AST(258.40 ± 90.80 U/L vs.114.33 ± 15.47 U/L)levels(P < 0.05).YQHX and FZHY administration can reverse the elevations of ALT(55.67 ± 17.91U/L,63.20 ± 27.60 U/L)and AST(138.50 ± 47.31 U/L,178.50 ± 42.24 U/L),in which,the decrease in AST was particularly significant compared with the CCl4 group(P < 0.05).And,no statistical significance was observed between the two intervention groups(P > 0.05).2.YQHX significantly improved the fibrosis in CCl4-induced cirrhosis rat model.Compared with the control group,the CCl4 group demonstrated obvious pathological features of cirrhosis: disruption of normal hepatic lobular structure,pseudolobular formation,increased inflammatory cells,and steatosis of some hepatic cells.Whlie YQHX and FZHY administration showed much thinner fibrous septum,and significantl increased pseudolobular volume compared with the CCl4 group.3.YQHX significantly increased the m RNA and protein expression levels of liver regeneration gene in cirrhosis rats.CK19-positive Hering tubes,known as ductular reaction,were observed in the CCl4 group,mainly located in the portal area.However,there were no significant increase in the intervention of YQHX and FZHY groups.The CCl4 group showed a significant decrease in the liver protein expression level of Cyclin D1 compared to the control group(P < 0.05),while YQHX administration markedly upregulated the expression level of Cyclin D1 compared to the CCl4 and CCl4 + FZHY groups(P < 0.05)according to the western blot test.The liver expression levels of Cyclin D1 detected by immunohistochemical showed the similar trend with the western blot results.In addition,the CCl4 group demonstrated lower expression levels of ki67 and PCNA compared to the control group(P < 0.05),YQHX and FZHY administration significantly increased the expression levels of ki67 and PCNA compared with the CCl4 group(P < 0.05),while the levels in the CCl4 +YQHX group were much higher than those in the CCl4 + FZHY group(P <0.05).Summary:1.YQHX can significantly improve the inflammation and fibrosis in CCl4-caused liver cirrhosis rat model;2.YQHX can significantly upregulate the liver expression levels of Cyclin D1,ki67 and PCNA,promote LR in cirrhosis rats,and improve liver inflammation and fibrosis.Conclusion:1.YQHX combined with ETV can significantly improve LR in patients with liver fibrosis caused by chronic hepatitis B,and significantly improved the liver fibrosis,which provides theoretical basis for the treatment of end-stage liver diseases;2.YQHX can significantly promote the proliferation of liver cells in rats after 2/3PH,reverse liver fibrosis by improving LR in cirrhosis rats model caused by CCl4;3.YQHX is expected to be a therapeutic agent for LR in cirrhosis,its definite mechanism remains to be further verified. |
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