Mechanisms Of Allium Victorialis L.Extracts On Liver Inflammation And Fibrosis

Objective:Liver fibrosis is the ultimate common results of chronic or repeated liver injury.Drugs,viruses,alcohol abuse,high fat,high sugar and malnutrition can lead to the occurrence of liver fibrosis.Hepatic stellate cells(HSCs)is the primary source of substrate composition,and it plays an important role in the process of the formation of liver fibrosis,while liver cells as the main target cells of various hepatotoxicity medium,when it is hited by a virus,the metabolites of alcohol and bile acid stimulation,it will release a large number of reactive oxygen species(ROS)and inflammatory mediators,and aggravate the occurrence of liver fibrosis.As a kind of food,AVL has been used in traditional Chinese medicine because of its anti-inflammatory,antioxidant,anti-cancer and other activities.In this study,alcohol,methionine choline deficiency(MCD)and thioacetamide(TAA)were used to establish hepatic fibrosis model in vivo,and AML 12,HepG2 and HSC cells were used to establish the corresponding in vitro,so as to study the potential mechanism of action of AVL extracts in fat accumulation,inflammatory response and hepatic fibrosis.Methods:(1)Ethanol extraction was used to prepare AVL extracts.(2)AVL ameliorated EtOH-Induced Liver Lipid Deposition and Inflammation.In vivo,C57BL/6 mice were randomly divided into six groups:normal group,ETOH group,ETOH+AVL(100 mg/kg,200 mg/kg and 300 mg/kg)and ETOH+silymarin(100 mg/kg).Mice in normal group were given liquid control diet every day,while mice in other group were given Lieber-DeCarli(L-D)containing 5%EOTH(vol/vol),at the same time the mice were treatmented with AVL(100 mg/kg,200 mg/kg and 300 mg/kg)and silymarin(100 mg/kg)for 4 weeks.At the end of the fourth week,all the mice except the normal group were given a single alcohol gavage(5g/kg).After the last gavage,all mice were sacrificed under anesthesia serum and liver samples were collected at-80℃.Serum ALT,AST and liver TG were detected.In addition,H&E staining,oil red staining and Nile red staining were used to observe the histopathological changes of the liver.Western blot and Q-PCR were used to detect the gene levels of proteins related to hepatic fibrosis,fat and inflammation.In vitro,AML 12 and HepG2 cells were treated with alcohol to simulate the model of fatty liver in vitro,cells were collected and detected by western blot analysis to determine the expression levels of nuclear receptors,SREBP1,Lipinl and Lipin2.(3)AVL modulated liver lipid deposition and inflammation methionine choline deficiency-induced fatty liver disease.Mice were randomly divided into six groups:normal group,MCD group,MCD+ AVL(100 mg/kg,200 mg/kg and 300 mg/kg)and MCD+silymarin(100 mg/kg).The control mice given MCS diet,while mice in other groups were given MCD diet,meanwhile the mice were administrated with AVL(100,200 and 300 mg/kg)and silymarin(100 mg/kg).After 4 weeks,all mice were sacrificed under anesthesia.Serum and liver were collected and stored at-80℃.In addition to the detection of serum ALT/AST and tissue TG,H&E staining was also used to observe the steatosis in liver tissues.Western blot and Q-PCR were used to detect the expressions of proteins related to fat synthesis,inflammation and fibrosis.In vitro,HepG2 cells were induced by OA to simulate the model of non-alcoholic fatty liver.At the same time,HepG2 cells were treated with AVL for 24h.After thses,cells were collected,and the western blot was performed to determine the expressions of proteins involved in nuclear receptor,fat synthesis.(4)AVL improved thioacetamide-induced hepatic inflammation and fibrosis in mice.ICR mice were randomly divided into six groups:normal group,TAA group,TAA+AVL(100,200 and 300 mg/kg)and TAA+silymarin(100 mg/kg).Except for the normal group,all mice in the other groups were given TAA by intraperitoneal injection for 5 weeks(first week:100mg/kg,3 times a week;second to the fourth week:200mg/kg,twice a week),and mice were administration with AVL and silyamrin by gavage every day.After 5 weeks,the mice were sacrificed under anesthesia.Blood and liver samples were collected for subsequent experiments.Serum AST/ALT levels were detected,then the effects of AVL on the histopathological changes of mouse liver was detected by H&E staining and Sirius Red staining,and then the expressions of proteins related to hepatic fibrosis,and inflammation in mice were detected by Western blot and immunohistochemical staining.In vitro,the supernatant medium of Raw 264.7 cells stimulated by LPS was used to stimulate HSC cells,the expressions of fibrosis indicators such as α-SMA and inflammatory factors such as IL-6 was detected to further confirm that AVL could improve liver inflammation and fibrosis.Results:(1)AVL extracts were stored at 4℃.(2)In alcoholic fatty liver model,AVL reduced the the levels of serum ALT/AST and liver TG increased by alcohol,improved the histopathological changes induced by alcohol,inhibited the expressions of SREBP1,CYP2E1,Lipinl and Lipin2.In addition,AVL regulated the expressions of nuclear receptors,such as PPARs,LXRs and FXR,reduced the releases of inflammatory factors(IL1α,IL1β,caspase1).AVL activated Sirt\AMPK\LKB1 expression,and inhibited the expressions of α-SMA,CollagenI and TIMP1.In vitro,AVL improved the formation of lipid droplets,regulated the expressions of SREBP1,Lipin1 and Lipin2,activated the expressions of LXRs and FXR,and reduced the releases of inflammatory factors in cells induced by alcohol.(3)In nonalcoholic fatty liver,methionine choline deficiency increased levels of liver TG and MDA,and serum AST/ALT,while AVL inhibited the expressions of these indicators.The results of histopathology,western blot and Q-PCR showed that MCD diet could lead to significant steatosis in liver,and even liver fibrosis at a certain extent.In addition,AVL also inhibited the expressions of proteins related to inflammation in MCD-induced mice,such as NLRP3,IL18 and IL1β.AVL regulated the expressions of proteins related to LKB1-AMPK signaling pathway and improved non-alcoholic fatty liver disease.AVL regulated the expressions of nuclear receptors,such as FXR,LXRs and PPARs.AVL inhibited the increases of hepatic fibrosis index,AVL inhibited the formation of lipid droplets in HepG2 cells induced by OA,which was related to the reduction of SREBP1 expression.In addition,the results of siAMPK showed that AVL regulated the LKB1-AMPK signaling pathway by regulating the expression of AMPK,activated the nuclear receptors(FXR,PPARs),inhibited the expressions of SREBP1 and reduced steatosis induced by OA.(4)In the thioacetamide-induced liver fibrosis model,AVL reduced the levels of serum AST and ALT in TAA-induced mice.The results of histopathology and western blot showed that AVL alleviated the collagen deposition in liver induced by TAA,inhibited the expressions of α-SMA,Collagen1,TIMP1,and inhibited the releases of inflammatory factors induced by TAA.In addition,AVL regulated the expressions of proteins related to the STAT3-TLR4 signaling pathway.In vitro,the results of western blot and Q-PCR showed that the AVL regulated the STAT3/TLR4 expressions and inhibited the activation of HSC cells induced by Raw supernatant,inhibited the expressions of inflammatory factors in the cells,such as IL6,NLRP3 and IL1R1.Conclusion:(1)AVL improved alcoholic fatty liver induced by alcohol.(2)AVL ameliorated methionine choline deficiency-induced nonalcoholic liver inflammation and fibrosis with steatosis;(3)AVL alleviated thioacetamide-induced liver inflammation and fibrosis.In conclusion,AVL has a certain inhibitory effect on liver inflammation and fibrosis induced by different factors in different species,and this study provides a theoretical basis for the development of anti-liver fibrosis drugs with the effects of clear targets,effective and safe.