Verapamil Ameliorates Hepatic Injury In Micewithnonalcoholic Fatty Liver Disease

Part One Protective effects of verapamil on nonalcoholic fatty liver disease induced liver injury in miceObjective To explore the protective effects of verapamil on non-alcoholic fatty liver disease(NAFLD)mice.Methods Male C57BL/6 mice were divided randomly into four groups,namely,control group(CON),verapamil group(VER),high fat diet group(HFD),and verapamil plus high fat diet group(VER+HFD).The CON group and VER group were feeding with normal diet while the HFD and VER+HFD group were feeding with 45%high fat diet.In the VER group and VER+HFD group,mice were injected intraperitoneally with 25mg/kg verapamil dissolved in 0.2ml sterile saline.In the CON group and HFD group,mice were treated in the same manner as that of the VER group,but they received saline instead of verapamil.After 10 weeks of feeding and 2 weeks of injection,the needed samples were collected.Histopathological alterations were observed under light-microscope,histologic scoring of the depth of tissue injury was performed according to the classical method;Total cholesterol(TC)and triglyceride(TG)were measured,alinine aminotransferase(ALT)and aspartate aminotransferase(AST)were evaluated,serum glucose and fasting insulin were analyzed.Results In the HFD group,the histological examination revealed significantly damaged hepatic tissues,with scattered fat drops,inflammatory cell infiltration;the hepatic histological score was significantly increased(P<0.05);the serum TG and TC,ALT and AST,blood glucose and fasting insulin level were significantly increased(P<0.05).These alterations were ameliorated after verapamil administration in HFD mice.Conclusion Verapamil treatment can atteunatehepatic injury and metabolic disorders in nonalcoholic fatty liver mice.Part Two Inhibiting effects of verapamil on hepatic Thioredoxin-interacting protein(TXNIP)/NOD-like receptors protein 3(NLRP3)inflammasome pathway in NAFLD mice.Objective To explore the inhibiting effects of verapamil on hepatic Thioredoxin-interacting protein(TXNIP)/nucleotide binding oligomerization domain(NOD)-like receptors protein 3(NLRP3)inflammasome pathway in NAFLD mice.Methods Male C57BL/6 mice were divided randomly into four groups,namely,control group(CON),verapamil(VER group),high fat diet group(HFD),and verapamil plus high fat diet group(VER+HFD).The CON group and VER group were feeding with normal diet while the HFD and VER+HFD group were feeding with 45%high fat diet.In the VER and VER+HFD groups,mice were injected intraperitoneally with25mg/kg verapamil dissolved in 0.2ml sterile saline.In the CON group and HFD group,mice were treated in the same manner as that of the VER and and VER+HFDgroup,but they received saline instead of verapamil.After 10 weeks of feeding and 2 weeks of verapamil treatment,the needed samples were collected.The pro-inflammatory factors IL-1? and IL-18 in hepatic issues were detected by enzyme-linked immuno sorbent assay;the expression of thioredoxin-interacting protein(TXNIP)/NOD-like receptors protein3(NLRP3)inflammasome were determined by Western blotting,real-time PCR and immunochemistry.Results The amount of pro-inflammatory factors IL-1? and IL-18 in hepatic tissue significantly increased(P<0.05);Increased TXNIP and NLRP3staining were observed in hepatic tissue and the protein and mRNA expressions of TXNIP,NLRP3,ASC and Caspase-lin heatic tissue increased after high fat diet.These alterations were ameliorated after verapamil administration in NAFLD mice.Conclusion Verapamil treatment can suppresseTXNIP mediated NLRP3 inflammasome activation in hepatic tissue,and reduce the hepatic inflamation.Part Three Inhibiting effects of verapamil on adipose Thioredoxin-interacting protein(TXNIP)/NOD-like receptors protein 3(NLRP3)inflammasome pathway in NAFLD mice.Objective To explore the inhibiting effects of verapamil on adipose Thioredoxin-interacting protein(TXNIP)/NOD-like receptors protein 3(NLRP3)inflammasome pathway in NAFLD mice.Methods Male C57BL/6 mice were divided randomly into four groups,namely,control group(CON),verapamil group(VER),high fat diet group(HFD),and verapamil plus high fat diet group(VER+HFD).The CON group and VER group were feeding with normal diet while the HFD and VER+HFD group were feeding with 45%high fat diet.In the VER and VER+HFD groups,mice were injected intraperitoneally with 25mg/kg verapamil dissolved in 0.2 ml sterile saline.In the CON group and HFD group,mice were treated in the same manner as that of the VER and VER+HFD groups,but they received saline instead of verapamil.After 10 weeks of feeding and 2 weeks of verapamil treatment,the needed samples were collected.The pro-inflammatory factors IL-1? and IL-18 in serum were detected by enzyme-linked immuno sorbent assay;the expression of thioredoxin-interacting protein(TXNIP)/NOD-like receptors protein3(NLRP3)inflammasome were determined by Western blotting and real-time PCR.Results The serum amount of pro-inflammatory factors IL-1? and IL-18 significantly increased(P<0.05);Increased TXNIP and NLRP3staining were observed and the expressions of TXNIP,NLRP3,ASC and Caspase-1 increased after high fat diet.These alterations were ameliorated after verapamil administration in NAFLD mice.Conclusion Verapamil treatment can suppresseTXNIP mediated NLRP3 inflammasome activation in adipose tissue,reduce the production of adipose tissue and inflammation.