| ObjectiveTo explore the curative effect,potential mechanism and functional metabolites of the patented Chinese medicine herbal composition“Longqi Jiangzhi decoction”(LQJZ)invented by Professor Fengbin Liu,a famous Chinese medicine doctor in Guangdong Province,on mice with non-alcoholic fatty liver disease(NAFLD).To explore the efficacy and mechanism of the main active ingredients of LQJZ and the main functional metabolites of LQJZ on NAFLD simultaneously.Altogether to provide scientific basic research support for LQJZ treatment of NAFLDMethods1.In order to clarify the effect of different dosage of LQJZ on the phenotype of NAFLD mouse model induced by Western Diet(WD),and to initially evaluate the efficacy of LQZJ on NAFLD,randomized grouping was carried out in stratified blocks based on initial body weight(n=10):normal control group(CON),WD group(sterilized purified water,20ml/kg/d),high-dose“Longqi Jiangzhi decoction”group(H-LQJZ,10.4g/kg/d),medium-dose“Longqi Jiangzhi decoction”group(M-LQJZ,5.2g/kg/d)and low-dose“Longqi Jiangzhi decoction”group(L-LQJZ,2.6g/kg/d).Except for the CON group,WD combined with high-sugar beverage formula(23.1g/L D-fructose and 18.9g/L D-glucose)ad libitum was used to establish the model.After the model was established to the 8th week,intragastric intervention was started,once a day,the intragastric intervention period is 4 weeks.During the period,the general condition and body weight changes of the mice were monitored and recorded.After the intervention,glucose tolerance test,insulin tolerance test and pyruvate tolerance test were carried out,the body weight growth rate,liver weight,liver index,white adipose tissue(WAT)weight,WAT index,blood sugar,serum alanine aminotransferase(ALT),aspartate Transaminase(AST),total triglyceride(TG),total cholesterol(TC),free fatty acid,insulin,homeostasis model assessment of insulin resistance(HOMA-IR)and quantitative insulin sensitivity check index(QUICKI)were measured,and pharmacodynamic evaluation of LQJZ by Hematoxylin and Eosin staining(HE staining)in liver tissue.2.In order to explore the difference of NAFLD phenotype and pathological degree induced by different dietary formula,and to evaluate the drug efficacy stability of LQJZ in different stages of NAFLD,randomized grouping was carried out in stratified blocks based on initial body weight(n=8):normal control group(CON),high-fat and high-cholesterol group(HFHC,sterilized purified water,10ml/kg/d),high-fat and high-cholesterol and“Longqi Jiangzhi decoction”(HFHC+LQJZ,2.6g/kg/d),western diet group(WD,sterilized purified water,1 0ml/kg/d)and western diet and“Longqi Jiangzhi decoction”group(WD+LQJZ,2.6g/kg/d).Except for the CON group,all mice were given high-sugar beverage formula and corresponding dietary formula ad libitum to establish models,and intragastric intervention was started from the first day of experiment,once a day,and the administration period was 12 weeks.During this period,the general condition and body weight changes of the mice were monitored and recorded.After the intervention,glucose tolerance test and insulin tolerance test were carried out,the body weight growth rate,liver weight,liver index,WAT weight,WAT index,serum ALT,AST,TG,TC,high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),tumor necrosis factor alpha(TNF-α),transforming growth factor beta(TGF-β)were measured,and the pathological results such as HE staining and oil red staining of liver tissue were performed for pharmacodynamic evaluation of LQJZ.3.Ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry(UHPLC-QE-MS)was conducted for the identification of active components in high-concentration LQJZ(520g/L)and low-concentration LQJZ(130g/L).We then import the original mass spectrometry data collected above into XCMS Online,and perform operations such as peak extraction,peak identification,peak integration,and retention time correction.Substance identification of peaks containing secondary fragment mass spectrometry data by comparing Biotree traditional Chinese medicine non-target metabolomics standard database and corresponding fragmentation law matching method.4.UHPLC-QE-MS was used to conduct non-targeted metabolomics detection on the liver tissue after LQJZ intervention in order to explore the functional metabolites with significant differences.On this basis,a series of bioinformatics analysis such as visual analysis of differentially functional metabolites and biological function annotation analysis were carried out,in order to clarify the potential pharmacodynamic mechanism of LQJZ in the treatment of NAFLD.Subsequently,the Q300 metabolic chip detection kit was used to perform absolute quantitative detection of the differentially fctional metabolites found in the non-target metabolomics results,and to verify the results of non-targeted metabolomics at the metabolite level.Finally,the potential mechanism pathways were verified at the transcriptional and protein levels by quantitative polymerase chain reaction(qPCR)and western blot(WB).5.A steatosis cell model was established by oleic acid and pamitic acid(OAPA)stimulation,and the PPARa inhibitor GW6471 was used to explore the effect of the differentially functional metabolite L-serine on the expression levels of proteins related to the PPARa signaling pathway.Oil red O staining,mitochondrial membrane potential kit and reactive oxygen species kit were used to detect the effect of L-serine on lipid deposition and mitochondrial function in steatogenic HepG2 cells.6.In order to explore the efficacy and mechanism of differentially functional metabolite L-serine in the treatment of NAFLD,randomized grouping was carried out in stratified blocks based on initial body weight(n=8):normal control group(CON),high-fat and high-cholesterol group(HFHC)and high-fat and high-cholesterol and L-serine group(L-Ser,100g/L ad libitum).Except for the CON group,all mice were given high-sugar beverage formula and high-fat and high-cholesterol diet formula ad libitum for constructing NAFLD model,and the intervention period was 8 weeks.During this period,the general condition and body weight changes of the mice were monitored and recorded.After the intervention,glucose tolerance test and insulin tolerance test were performed,the body weight growth rate,liver weight,liver index,WAT weight,WAT index,serum ALT,AST,TG,TC,HDL-C,LDL-C,TNF-α were measured,and the pathological results such as HE staining and oil red staining of liver tissue were performed for pharmacodynamic evaluation of L-serine.Quantitative polymerase chain reaction(qPCR)and WB were used to verify the effect of L-serine on PPARα signaling pathway.Results1.Body weight,body weight growth rate,Lee’s index,liver weight,liver index,WAT weight,WAT index,blood glucose,ALT,AST,TG,TC,insulin,HOMA-IR,free fatty acid were significantly increased in WD diet-induced NAFLD mice(P<0.05),and QUICKI decreased significantly(P<0.05).Glucose tolerance test and insulin tolerance test showed that the mice in the WD group had impaired fasting blood glucose,abnormal glucose tolerance and insulin resistance,and hepatic HE staining showed diffuse hepatocytes,loose cytoplasm,impaired the normal structure of liver lobules,and disordered arrangement of liver cells.Intervention of various doses of LQJZ could delay the body weight growth rate of NAFLD mice,reduce liver weight,WAT weight,WAT index,ALT,AST,TG,TC,insulin,HOMA-IR,free fatty acid levels(P<0.05),and increase QUICKI(P<0.05),glucose tolerance test and insulin tolerance test suggested that LQJZ could improve impaired fasting blood glucose,abnormal glucose tolerance and insulin resistance.HE staining showed that the pathology of the liver after LQJZ intervention was improved,the structure of the hepatic lobule was clearer than that of the WD group,diffusely distributed tiny lipid droplets could be seen in the liver tissue,and no inflammatory cell infiltration was seen in the portal area.2.Both HFHC diet and WD diet can successfully induce the NAFLD mouse model,but from the liver weight,liver index,WAT weight,WAT index,and pathological results,it can be intuitively found that the liver volume and mass of the NAFLD model induced by HFHC diet are higher than those induced by WD diet.The white fat accumulation of NAFLD model induced by WD diet was heavier than that induced by HFHC diet(P<0.05).In terms of liver pathology,the HFHC diet-induced NAFLD model showed more extensive and more obvious lobular inflammation and ballooning than the WD diet-induced NAFLD model.LQJZ has good pharmacological effects on both feed-induced NAFLD mouse models,and can significantly reduce body weight,liver weight,WAT weight,WAT index,ALT,TG,TNF-α,TGF-β induced by HFHC diet or WD diet increased(P<0.05),while improving impaired fasting blood glucose,impaired glucose tolerance and insulin resistance(P<0.05),LQJZ can significantly reduce the increase in body weight growth rate,Lee’s index,TC and LDL-C induced by HFHC diet(P<0.05).Both HE staining and oil red O staining indicated that LQJZ could significantly ameliorate the degree of liver inflammation and fat deposition induced by HFHC diet or WD diet.3.The positive and negative ion flow chromatograms of the high-concentration and low-concentration LQJZ prepared in different batches have the same shape trend,and the overall peaks of the components detected at different retention times are highly consistent,suggesting that the types of compound components contained in the high and low concentration of LQJZ are highly consistent.Screening according to the CompositeScore threshold(>0.6),298 active ingredients can be identified in positive ion mode,and 180 active ingredients can be identified in negative ion mode.There are 26 active ingredients detected jointly by the two modes,including gentiopicroside,hesperidin,isovitexin,swertenin,astragalus isoflavane,and alisitol C-23-acetate,naringenin,naringin-chalcone,naringenin-7-O-glucoside,quercetin-3-arabinoside,isorhamnetin,etc.The types of compounds covered mainly include flavonoids,terpenes,Triterpenes,iridoids,alkaloids,etc.The response values of these active ingredients are sorted,and the higher the response value,the higher the relative content of the active ingredient in the LQJZ.In the negative ion mode,the response values of the five active ingredients,gentiopicroside,hesperidin,swertigin,formononetin,and melissain,were relatively high.In the positive ion mode,the response values of the five active ingredients such as swertoside,mellowin,naringin-chalcone,naringenin-7-O-glucoside,and gentiopicroside were significantly higher than those of other active ingredients.4.Both principal component analysis and orthogonal projections to latent structuresdiscriminant analysis in non-target metabolomics showed that the overall metabolite levels of HFHC+LQJZ group and HFHC group were significantly different.Differential metabolite analysis,matchstick nalysis and hierarchical cluster analysis found that L-serine(L-Ser),eicosapentaenoic acid(EPA),docosahexaenoic acid(DHA),etc.are potential differential functional metabolites,among which L-Ser is the differentially functional metabolite that jointly detected in positive and negative ion mode simultaneously.KEGG enrichment analysis and differential abundance analysis of differentially functional metabolites suggested that the pharmacodynamic mechanism of LQJZ may be related to glycine-serine-threonine metabolism and PPAR signaling pathway.Targeted metabolomics found that the content of D-fructose and D-glucose in the liver of mice in the HFHC group was significantly higher than that in the CON group(P<0.05),and the content of D-fructose and D-glucose in the liver of mice after LQJZ intervention Both decreased(P<0.05).The contents of glycine,L-threonine,L-Ser,EPA,and DHA in the liver of mice in the HFHC group were significantly lower than those in the CON group(P<0.05),and these five functional metabolites in the liver of mice after LQJZ intervention were significantly increased(P<0.05).The relative mRNA and protein expressions of PPARα,CPT and ACOX in the liver of mice in the HFHC group were significantly lower than those in the CON group(P<0.05),and the relative mRNA and protein expressions of PPARa and CPT in the mouse liver were significantly increased after LQJZ intervention(P<0.05),the relative protein expression of ACOX was significantly increased(P<0.05),the results showed that LQJZ can increase the transcription level and protein expression level of PPARα pathway-related genes in the liver tissue of NAFLD mice.5.The results of CCK8 suggested that 24 hours of L-Ser intervention(0-256μM)had no significant effect on the cell viability of HepG2 cells(P>0.05),but 48 hours of L-Ser intervention(32-256μM)significantly inhibited the viability of HepG2 cells(P<0.05).Both 24-hour GW6471 intervention(8-64μM)and 48-hour GW6471 intervention(4-64μM)significantly inhibited the viability of HepG2 cells(P<0.05).The results of WB showed that after OAPA intervention for 24 hours,the expressions of PPARα and its downstream proteins CPT1 and ACOX were down-regulated,and after the intervention of different concentrations of L-Ser(50μM,100μM and 250μM),the protein expression of PPARα,CPT1 and ACOX were up-regulated.Intervention of PPARα inhibitor GW6471 alone in HepG2 cells for 24 hours can lead to a decrease in the protein expression levels of CPT1 and ACOX,in which the protein expression of ACOX was significantly down-regulated(P<0.05),but GW6471 could not significantly inhibit the up-regulation of CPT1 and ACOX proteins by L-Ser(P>0.05),indicating that L-Ser can activate the PPARα signaling pathway,but not in an independent manner.6.The body weight,weight growth rate,Lee’s index,liver weight,liver index,and WAT weight of NAFLD mice induced by HFHC diet were significantly increased(P<0.05),and serum ALT,AST,TC,LDL-C,and TNF-α were significantly increased(P<0.05),glucose tolerance test and insulin tolerance test suggested that 8 weeks of HFHC dietary intervention lead to abnormal glucose tolerance and postponed insulin release,but the sensitivity of cells responding to insulin has not been completely damaged,liver HE staining and Oil red O staining indicated fatty liver-related pathological changes.Supplementation of L-Ser can postpone the body weight growth rate of NAFLD mice,reduce liver weight,liver index,ALT,AST,TNF-α(P<0.05),improve liver pathology,but cannot significantly reduce WAT weight,TC,LDL-C,and the improvement of abnormal glucose tolerance in NAFLD mice was not obvious.After L-Ser intervention,the relative mRNA and protein expressions of PPARα and CPT in the mouse liver were significantly increased(P<0.05),and the relative protein expression of ACOX was significantly increased(P<0.05).The results indicated that L-Ser can improve transcription levels and protein expression levels of PPARα pathway-related genes in liver tissues of NAFLD mice.Conclusion1.The NAFLD mouse model induced by WD diet and HFHC diet can lead to different stages of NAFLD,The NAFLD mouse model induced by WD diet is more inclined to early non-alcoholic fatty liver,while the NAFLD mouse model induced by HFHC diet is more inclined to non-alcoholic steatohepatitis(NASH).2.LQJZ can improve the degree of obesity,glucose and lipid metabolism disorder,insulin resistance,inflammatory state,and liver fat deposition in NAFLD mice induced by both WD diet and HFCH diet,and the drug effect is more obvious in NASH mice.3.Gentiopicroside,swertigin,hesperidin and melissain are the main active ingredients in LQJZ that exert the pharmacological effects of lowering lipid and protecting liver.4.Metabolomics confirmed that LQJZ mainly plays a role in the treatment of NASH by up-regulating the PPAαR signaling pathway.L-Ser,glycine,EPA,DHA are the potential differentially functional metabolites of LQJZ in the treatment of NASH,among which L-Ser is one of the main functional metabolites.5.Supplementation of L-Ser can improve inflammatory injury in NAFLD mice,and its mechanism is also related to PPARα signaling pathway. |
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