The Intervention Study Of Shengjiang Powder On Non-alcoholic Fatty Liver Disease In Rats

Objectives:To observe the intervention of Chinese medicine compound Sheng jiang san on nonalcoholic fatty liver disease induced by highfat diet in rats and its preliminary mechanism.Methods: Twelve of 36 male Sprague-Dawley rats were randomly selected as the normal control group,the basic diet was given,the rest as the high-fat diet group,the high-fat diet was given(79% basal diet + 10% egg yolk powder + 10% lard + 1% cholesterol).At the end of 4 weeks,6 rats were randomly sacrificed in each group.Hepatic tissue was stained with hematoxylin-eosin(HE)and oil red O to observe the fatty degeneration of hepatocytes,evaluating the modeling progress.After successful modeling,except for the normal control group(NOR),the rats in the high fat diet group were randomly divided into nonalcoholic fatty liver disease(NAFLD)group,Sheng jiang san(SJS)group and fenofibrate(FNBT)group,6 in each group.SJS group was given SJS(No decoction: 225 mg/kg/d of bombyx batryticatus,104 mg/kg/d of periostracum cicada,375 mg /kg/d of curcuma longa,333 mg/kg/d of rhubarb)by intragastric gavage,FNBT group was given fenofibrate suspension(100 mg/kg/d),NOR group and NAFLD group were given equal volume of saline,1/day for 4 weeks.Observe the general situation of body weight,activity,food intake in the entire modeling and dosing period;All the rats were sacrificed at the end of the 4th week.Serum triglyceride(TG),total cholesterol(TC),alanine aminotransferase(ALT),aspartate aminotransferase(AST)and TG、TC level in fresh liver were taken.Liver tissues were stained with HE and Oil Red O to observe the histological changes such as hepatic steatosis;Immunohistochemistry was used to detect the expression of phosphorylated AMP-activated protein kinase alpha(p-AMPKα)in liver tissue.Western blot was used to detect the expression level of AMPKα,p-AMPKα,mammalian target of rapamycin(mTOR)and p-mTOR.Results: 1.General situation: Compared with the NOR group,NAFLD model group rats decreased activity,slow response,body weight increased significantly(P <0.05);After drug intervention,the activity and reactivity of rats improved to a certain extent,the body weight decreased significantly(P <0.05);Compared with NOR group,serum TG level increased(P <0.05)and hepatic TG and TC levels increased significantly in NAFLD group(P <0.01),hepatic cord disorderly arranged,hepatocellular swelling,a large number of lipid deposition in the cytoplasm of hepatocytes,severe hepatic steatosis appeared.The level of serum TG and the content of TG and TC in liver of SJS group were significantly decreased after drug intervention(P <0.05),and the hepatic cord recovered radially arranged structure,the size of hepatocyte was normal,the amount of lipids in hepatocyte decreased significantly,hepatic steatosis was significantly improved,the above indicators of FNBT group also showed varying degrees of recovery.3.Mechanism: Compared with NOR group,the protein level of p-AMPKαin liver tissue of NAFLD group was significantly decreased and the protein level of pmTOR was significantly increased;After drug intervention,p-AMPKα expression was significantly up-regulated and p-mTOR was significantly decreased in SJS group,p-AMPKα protein level was significantly up-regulated in FNBT group,and p-mTOR protein level was not significantly changed.Conclusion: 1.Sheng jiang san can ameliorate hepatic steatosis and serum lipid in high-fat-diet induced nonalcoholic fatty liver disease rats,which has a significant therapeutic effect;2.Sheng jiang san may play an anti-NAFLD effect by regulating AMPK / mTOR pathway,inhibiting triglyceride synthesis and reducing hepatic lipid deposition.Objectives : Using the CRISPR / Cas(clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins)technology,p X330-Pten plasmid was injected into the blood circulation of SD rats through the tail vein to edit the Pten(phosphate and tension homology deleted on chromsome ten)in rat liver,establish a non-alcoholic fatty liver disease(NAFLD)rat model and to observe the effect of Sheng jiang san on NAFLD rat model induced by Pten knock-out in liver.Methods: 1.Optimal p X330-Pten dose and modeling time required for NAFLD rat model: Sixty male Sprague Dawley rats were randomly divided into four groups: control group(NOR),p X330 vector(VECTOR),p X330-Pten low-dose group(LD),p X330-Pten medium-dose group(MD),p X330-Pten high-dose group(HD),twelve rats in each group.LD,MD and HD group were injected with p X330-Pten plasmid of 75 ug/100 g,150 ug/100 g,300 ug/100 g respectively.VECTOR group was injected with 150 ug/100 g p X330 vector,NOR group was injected with an equal volume of saline,the injection volume is10 ml,the injection was finished in 30 seconds.At 6 and 9 weeks after injection,6 rats were sacrificed in each group,and the contents of TG,TC,ALT and AST in serum were measured.The liver tissues were stained with HE staining and oil red O to observe the histological changes such as steatosis.DNA was extracted from fresh liver tissue and deep sequencing was used to detect the efficiency of target mutation around Pten locus.Quantitative real-time reverse transcription PCR was used to detect the m RNA expression level of Pten.Western blot was used to detect the protein levels of Pten,phosphorylated protein kinase B(p-Akt).2.To investigate the effect of Shengjiang san on NAFLD rats induced by liver Pten knockout: Twenty-four male Sprague-Dawley rats were randomly divided into four groups: normal control group(NOR),model group(p X330-Pten),Sheng jiang san group(SJS),fenofibrate group(FNBT),6 rats in each group.p X330-Pten group,SJS group and FNBT group were injected with 300 ug / 100 g p X330-Pten plasmid through tail vein,NOR group injected with equal volume of saline,the injection volume was 10 ml,and injection was completed in 30 seconds.At the end of the 6th week after injection,SJS group was given SJS(No decoction: 225 mg/kg/d of bombyx batryticatus,104 mg/kg/d of periostracum cicada,375 mg /kg/d of curcuma longa,333 mg/kg/d of rhubarb)by intragastric gavage,FNBT group was given fenofibrate suspension(100 mg / kg / d),NOR group and p X330-Pten group was given an equal volume of saline,1/day,for 4 weeks.Observe rat body weight,activity,food intake during the experiment.All the rats were sacrificed at 4 weeks after the administration,and the serum levels of TG,TC,ALT and AST were measured;HE stain and oil red O stain was performed to observe the pathological changes in rat liver.Results: 1.NAFLD rats modeling: Compared with the NOR group,with the increase of the dosage of p X330-Pten plasmid and the time of modeling,the liver lipid deposition in LD group,MD group and HD group increased gradually,and the body weight and blood lipid had not changed significantly after 6 weeks of high dose feeding,but a large number of lipid deposited in rat liver,suggesting that a rat model of early stage NAFLD has been successfully established;At 9 weeks after high-dose modeling,the body weight of rats increased significantly(P <0.05),serum TG increased significantly(P <0.05)and the liver showed more severe lipid deposition;The genomic DNA insertion loss frequency of hepatic tissue was increased in HD group,Pten m RNA and protein levels were significantly decreased(P <0.05)and P-Akt protein level was significantly increased(P <0.05);2.Effects of Shengjiang San on NAFLD rats induced by liver Pten knockout: Compared with the NOR group,the activity of the p X330-Pten group was decreased,the response was slow and the body weight was significantly increased(P <0.05),and no significant recovery was found after the drug intervention;In terms of lipid levels and liver pathology: Compared with the NOR group,the serum TG of p X330-Pten group was significantly increased(P <0.05),and there was a large amount of lipid deposition in the liver.No significant amelioration was found in serum TG level and hepatic steatosis after drug intervention.Conclusion:1.Based on the CRISPR / Cas system,300 ug/100 g of p X330-Pten plasmid was delivered into the blood circulation of rat by tail vein injection in one time,and Pten gene was effectively edit in rat liver after 6 weeks,nonalcoholic fatty liver disease was successfully established.2.Sheng jiang san has no significant treating effect on NAFLD rats induced by liver Pten down regulation.