| Aim:The incidence rate of nonalcoholic fatty liver disease(NAFLD)is higher than that of premenopausal women.The protective effect of estrogen or phytoestrogens on hepatic steatosis has been widely confirmed.Xiao Yao San(XYS),as a classic prescription with the effect of soothing the liver,is widely used in the clinical practice of traditional Chinese medicine to treat various symptoms of menopause.Pharmacological studies have found that XYS has an estrogen like effect and can enhance the expression of ERa mRNA in perimenopausal rat ovaries.In addition,XYS can significantly improve abnormal lipid metabolism.However,whether XYS can improve postmenopausal hepatic steatosis has not been reported.At the same time,there is no consensus on the common TCM syndrome types of postmenopausal NAFLD.Based on the above research background,this topic intends to collect clinical medical records to explore the distribution of NAFLD among different TCM Syndromes of postmenopausal syndrome;The pharmacological effects of XYS on NAFLD in postmenopausal women were investigated by animal and cell experiments,and the molecular mechanism was elucidated by LXRα/SrebP-1C pathway mediated by hepatic estrogen receptor α(ERα).Mehtod:1.Theoretical discussion:Systematic literature retrieval was conducted to summarize the understanding of ancient Chinese medicine book■s and modern scholars on the disease name,etiology,pathogenesis and treatment of menopause syndrome.Analysis of the main pathogenesis of postmenopausal NAFLD is kidney deficiency,liver stagnation,phlegm and stasis in the liver.Liver conditioning is an important treatment for postmenopausal NAFLD,which provides theoretical basis for XYS treatment of postmenopausal hepatic steatosis.To discuss the relationship between menopause and NAFLD from the perspective of modern medicine.2.Clinical data study:the cross-sectional study method was used.200 patients with postmenopausal syndrome who met the inclusion criteria were collected from the menopause clinic,gynecology clinic and traditional Chinese medicine gynecology clinic of Huai an Women’s and children’s Hospital,and the correlation between influencing factors and syndrome types was analyzed;Objective to investigate the prevalence rate of NAFLD and the distribution pattern of TCM syndromes in menopausal syndrome patients,and to identify the common TCM Syndromes of NAFLD after menopause.3.Animal experimental research:(1)XYS was prepared and the contents of paeoniflorin,paeoniflorin,Bupleurin A and bupleurin D were determined,and the quality of XYS was evaluated.(2)Replication,grouping and administration of postmenopausal NAFLD mouse model:ApoE-/-mice meeting the experimental requirements were randomly divided into control group,model group,estrogen group,XYS high-dose group and XYS low-dose group,with 10 mice in each group.The control group was fed with normal feed after sham operation.The other groups were fed with high fat diet after both ovaries were excised under anesthesia.(3)Effects of XYS on body weight,plasma TG,ALT and NEFA of climacteric NAFLD model:Body weight,fasting plasma triglyceride(TG),alanine aminotransferase(ALT)and neutral fatty acid(NEFA)levels of mice in each group were detected.(4)Effects of XYS on liver weight,TG and hepatic steatosis in menopausal NAFLD model:Liver weight and TG content of mice in each group were detected,lipid content was measured by HE and oil-red O staining,and lipid droplets were observed under transmission electron microscopy.(5)The mRNA levels of SREBP-1C,FAS,ACC1,SCD-1,LXRα,PPARα,CPT1 and ACOX1 were analyzed by RT-qPCR.The expression of LXRa and SREBP-1C protein in liver was detected by Western blot.ERa protein expression was examined by Western blot and immunostaining.4.Cell experimental study:(1)Preparation of XYS-serum:30 mice were randomly divided into control group,XYS-high-dose group and XYS-low-dose group,with 10 mice in each group.Drugs were given in different groups.The drug was administered for 7 consecutive days.Blood was collected from orbit 1 hour after the last administration,followed by centrifugation for 10 minutes at 1500rp/min.Serum was separated in a sterile environment,inactivated by 56℃constant temperature water bath,and filtered by a 0.22μm filter.Finally,XYS-H serum,XYS-L serum and blank control serum were obtained.(2)L02 cell culture:L02 cells were cultured in DMEM medium supplemented with 10%fetal bovine serum(100 U/mL penicillin+100 μg/mL streptomycin)at 37℃ and stored in 5%CO2 incubator.Cells are digested and subcultured by trypsin,and 2-5 subgenerations are used in the experiment.(3)Hepatic steatosis cell model was established,and then grouped and administered:L02 cells at logarithmic growth stage were cultured in 6-well plates for 24h,which were divided into 5 groups:control group,model group,XYS-H serum,XYS-L serum and estrogen group.To induce steatosis,cells in all groups except the control group were exposed to a mixture of 1 mmol/L FFA(Oleate:Palmitate=2:1)for 24h,then administered separately and cultured for another 24h.(4)Cell activity test:the cells were treated with blank control serum,XYS-H serum and XYS-L serum at a final concentration of 10%for 24h,respectively,and MTT test was performed to evaluate the cell activity.Untreated cells were used as controls.(5)Effects of XYS-serum on mRNA and protein expression of genes related to lipid metabolism in L02 cells:The mRNA expression of LXRα was analyzed by RT-qPCR.The protein expression of LXRa was detected by Western blotting.The mRNA expression of SREBP-lc was analyzed by RT-qPCR.The protein expression of SREBP-lc was analyzed by immunofluorescence staining.The mRNA expressions of FAS,ACC1 and SCD-1 were analyzed by RT-qPCR.(6)Effects of XYS-serum on ERα expression and activity of L02 cells:The mRNA expression of ERa was analyzed by RT-qPC R.ERa activity was detected by double luciferase reports.The expression of ERa protein was analyzed by immunofluorescence staining.(7)Inhibition of XYS-serum on L02 cell steatosis mediated by ERa signaling pathway:Effects of estrogen receptor inhibitor ICI 182,780 on the reduction of lipid accumulation and TG content in L02 cells;Whether ICI 182,780 reversed the effects of XYS-serum on the mRNA levels of LXRa and SREBP-1C were analyzed by RT-qPCR.Whether ICI 182,780 reversed the mRNA expression of FAS,ACC1 and SCD1 on Xys-SERUM was analyzed by RT-qPCR.5.Perform data analysisResults:1.Comparison of general information:Except MHT supplementation,there was no significant difference between patients(P>0.05),and the other factors were distributed differently among patients(P<0.01).2.The distribution of TCM syndromes in patients with menopausal syndrome is as follows:The distribution was different(P<0.05).Liver and kidney yin deficiency syndrome(32%)>kidney deficiency and liver depression syndrome(27.5%)>heart kidney Disharmony Syndrome(24.5%)>kidney yin and yang deficiency syndrome(16%).3.Correlation analysis between TCM Syndrome Types and general factors of postmenopausal syndrome:The distribution of TCM syndromes in the course of disease,BMI,age,education level,MHT supplement,exercise,whether there were hypertension or diabetes mellitus were different(p<0.05 or p<0.01).There was no significant difference in the distribution of TCM Syndrome Types in K score,postmenopausal stage,menopausal mode,smoking and drinking(P>0.05)4.the incidence rate of NAFLD in menopausal syndrome:There were 200 patients with menopausal syndrome who met the inclusion criteria,including 26 patients(13%)who met the diagnostic criteria of NAFLD.5.Correlation analysis of influencing factors of NAFLD after menopause:Apart from hypertension,there was no significant difference between the patients(p>0.05).The other factors included age,postmenopausal stages,BMI,menopause,whether MHT treatment,whether smoking,drinking,exercise or diabetes had different distribution characteristics among patients(p<0.05 orp<0.01).6.Distribution of NAFLD syndrome types after menopause:There was significant difference in the distribution of four different syndrome types of NAFLD after menopause(P<0.01).The rate of NAFLD in kidney deficiency and liver depression type was significantly higher than that in other syndrome types.7.Quality control and composition research of XYS:The chromatographic peaks of paeoniflorin,paeoniflorin,saikosaponin a and saikosaponin d in XYS extract were identified and determined by HPLC.The contents were 1.26mg/g,2.58mg/g,1.33mg/g and 1.08mg/g respectively.8.Effects of XYS on body weight,plasma TG,ALT and NEFA in menopausal NAFLD model:XYS had no effect on the body weight of Ovx/ApoE-/-mice(P>0.05).The plasma levels of TG,ALT and NEFA in model group were significantly increased(P<0.01).However,the levels of TG,ALT and NEFA in plasma were significantly decreased after xiaoyao SAN treatment(P<0.05 or P<0.01).9.Effects of XYS on liver weight,TG and hepatic steatosis in menopausal NAFLD model:Compared with the control group,liver weight and liver triglyceride content in the model group were significantly increased(P<0.01),while compared with the model group,high dose XYS group(13.0g/kg),low dose XYS group(6.5g/kg)and E2 group,Liver weight and triglyceride content were significantly decreased(P<0.05 or P<0.01).The severity and lipid content of liver steatosis in model group were significantly increased(P<0.01).XYS could significantly reduce liver lipid accumulation and liver cell steatosis in a dose-dependent manner(P<0.05).10.Effects of XYS on lipid metabolism genes and ERa expression in climacteric NAFLD model:In model group,the mRNA expression of TG synthesis related genes(including SREBP-lc and its downstream genes FAS,ACC1 and SCD-1)was significantly increased(P<0.01),and the mRNA expression of PPARα and its target genes ACOX1 and CPT1)involved in fatty acid β oxidation were significantly decreased(P<0.05).XYS treatment significantly down-regulated the mRNA levels of liver TG synthesis related genes(P<0.05 or P<0.01),but had no effect on the mRNA expressions of PPARα,ACOX1 and CPT1(P>0.05).The protein expression levels of LXRa and SREBP-1C were significantly increased in the model group(P<0.01),while XYS decreased the protein levels of LXRa and SREBP-1c in a dose-dependent manner(P<0.01 or P<0.05).11.XYS up-regulates the expression of ERa in liver of climacteric NAFLD mice:Immunohistochemical and Western blot analysis showed that the level of ERa protein in the liver of model group was significantly decreased(P<0.01),while XYS treatment significantly inhibited the down-regulation level of ERα protein in the liver of menopausal NAFLD mice(P<0.01 or P<0.05).12.Effects of XYS-serum on Steatosis and ALT activity of L02 cells:The results of MTT assay showed that XYS-H serum and XYS-L serum had almost no cytotoxicity.13.Effects of XYS-serum on FFA-induced steatosis and ALT activity of L02 cells:Compared with the control group,cells in the model group treated with FFA showed significant lipid accumulation(P<0.01),and the number of lipid droplets in the Xys-serum group was significantly decreased(P<0.05).Similarly,TG content and ALT activity in XYS-serum group were significantly decreased in steatosis cells(P<0.05).The content of TG and ALT activity in FFA group were significantly higher than those in control group(P<0.01).14.Effects of XYS-serum on mRNA and protein expression of Lipid-metabolism-related genes in L02 cells:The mRNA and protein levels of LXRa in FFA group were significantly increased(P<0.01),and the expression of LXRa in XYS-serum was significantly decreased(P<0.05).In addition,XYS-serum inhibited the mRNA levels of SREBP-1c and its target genes(FAS,ACC1 and SCD-1)(P<0.05).Immunofluorescence analysis showed that the change trend of SREBP-lc protein expression in different groups was consistent with that of mRNA expression.15.Effects of Xys-serum on ERa expression and activity of L02 cells:Compared with the control group,Xys-serum significantly increased ERα mRNA level(P<0.05).Similarly,immunofluorescence staining also showed that XYS-serum significantly increased the expression of ERα protein in L02 cells.The results showed that XYS-serum significantly increased the activity of ERa luciferase(P<0.01).16.Inhibitory Effect of XYS-serum on L02 cell steatosis mediated by ERa signaling Pathway:After the interference of estrogen receptor inhibitor ICI 182,780 on ERα in L02 cells,oil red O and Nile red staining showed that the two ICI182,780 intervention groups showed significant lipid accumulation compared with the control group.(P<0.01)eliminated the inhibitory effect of XYS-Hserum on FFA-stimulated lipid accumulation in L02 cells.Similarly,the inhibition of XYS-Hserum on intracellular TG content was partially eliminated by ICI 182,780.The combination of XYS-Hserum with ICI182,780 significantly reduced the activity of ER luciferase(P<0.01).ICI 182,780 partially reversed the inhibition of XYS-Hserum on the mRNA levels of LXRa,SREBP-1c and their target genes in FFA-stimulated L02 cells.Conclusions:The syndrome of kidney deficiency and liver stagnation is a common TCM syndrome type of postmenopausal NAFLD.XYS can inhibit hepatic steatosis in postmenopausal NAFLD mouse model and FFA-induced L02 cell model.The mechanism may be related to inhibiting the expression of transcription factor SREBP-lc by regulating liver ERα,and then inhibiting the expression of downstream hepatic lipid metabolic factors(FAS,ACC1,SCD-1)of SREBP-lc.Therefore,XYS can be used to prevent NAFLD-related lipid metabolism diseases in postmenopausal women. |
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