Effect Of Different Doses Of Vitamin C And Ursodeoxycholic Acid On High-fat-diet-induced NAFLD In Mice

Part Ⅰ Prophylactic and therapeutic effects of different dose of vitamin C on non-alcoholic fatty liver disease in mice ObjectiveThe aims of this study were to investigate the prophylactic and therapeutic effects of different doses of vitamin C(Vit C)on non-alcoholic fatty liver disease(NAFLD)in high-fat-diet-fed mice.Methodssix-eight week-old male C57BL/6J mice were randomly divided into early prophylactic and late therapeutic groups.Both of these experimental processes had five subgroups(six mice per subgroup): control subgroup,high-fat diet subgroup,low-dose Vit C(15 mg/kg per day)subgroup,medium-dose Vit C(30 mg/kg per day)subgroup and high-dose Vit C(90 mg/kg per day)subgroup.In the early prophylaxis,control subgroup were given normal chow and normal saline,high-fat subgroup were given high-fat diet(HFD)and equal-dose normal saline,Vit C-administered subgroups were given high-fat diet and different doses of Vit C,all mice were prophylactically administered for 12 weeks.In the late therapy,mice were fed either a normal chow or an HFD for 6 weeks.Upon confirmation of steatosis,the HFD-fed mice were assigned to high-fat and Vit C-administered subgroups.Vit C-administered subgroups were treated with different doses of Vit C for another 12 weeks,control and high-fat subgroups were treated with equal volume of NS as a vehicle for 12 weeks.Body mass,liver wet weight,liver index,perirenal adipose tissue mass weight,homeostasis model assessment of insulin resistance(HOMA-IR),serum index [ALT,AST,total cholesterol(TC),triglyceride(TG),blood glucose(Glu)] level s,and HE and Oil Red O staining histopathological analysis were observed.ResultsIn the early prophylaxis,mice in high-fat subgroup showed significantly increased body weight,liver wet weight,liver index,perirenal adipose tissue mass weight,Glu,TG and TC levels compared with the control subgroup mice(P < 0.05),and mice in high-fat subgroup exhibited distinct liver steatosis(P < 0.05);the lowered body weight and perirenal adipose tissues mass weight were observed in low-dose and medium-dose Vit C subgroups compared with high-fat subgroup(P < 0.05),and liver steatosis was also significantly improved(P < 0.05);the higher body weight and more severe liver steatosis were observed in high-dose Vit C subgroup compared with medium-dose Vit C subgroup(P < 0.05).In the late therapy,the body weight and perirenal adipose tissues mass weight in medium-dose Vit C subgroup were lower than those in high-fat,low-dose Vit C and high-dose Vit C subgroups(P < 0.05),whilst the liver steatosis in medium-dose Vit C subgroup was improved compared with high-fat subgroup(P < 0.05);the higher body weight,liver wet weight,perirenal adipose tissues mass weight and more severe lobular inflammation were observed in high-dose Vit C subgroup compared with high-fat subgroup(P < 0.05),meanwhile mice in high-dose Vit C subgroup showed higher ALT and AST levels,and liver steatosis,ballooned hepatocytes and lobular inflammation than medium-dose Vit C subgroup(P < 0.05).Whether in the early prophylasix or in the late therapy,the TG level in low-dose Vit C subgroup was lower than that in high-fat subgroup(P < 0.05),while both the Glu and TG level in medium-dose Vit C and high-dose Vit C subgroups were lower than those in high-fat subgroup(P < 0.05).The lowered perirenal adipose tissues mass weight was observed in high-fat subgroup in the early prophylasix compared with high-fat subgroup in the late therapy(P < 0.05);the lowered body weight,perirenal adipose tissues mass weight and improved liver steatosis,ballooned hepatocytes and lobular inflammation were observed in low-dose Vit C subgroup in the early prophylasix compared with low-dose Vit C subgroup in the late therapy(P < 0.05);and the lowered body weight,liver weight,perirenal adipose tissues mass weight,ALT and AST levels and improved liver steatosis,lobular inflammation were observed in high-dose Vit C subgroup in the early prophylasix compared with high-dose Vit C subgroup in the late therapy(P < 0.05).ConclusionsSupplementation of low-and medium-dose Vit C can early protect HFD-fed mice from the development of NAFLD,meanwhile medium-dose Vit C can improve the metabolic changes and liver steatosis induced by HFD in mice;low-dose Vit C showed no improvement in the therapy of NAFLD;however,high-dose Vit C for the management of NAFLD was risky whether in early prophylaxis or late therapy,especially for the late therapy,high-dose Vit C can aggravate NAFLD progress to severe phenotype.Part Ⅱ Co-administration of medium-dose vitamin C and ursodeoxycholic acid slowed the development of non-alcoholic fatty liver disease in HFD-fed mice ObjectiveThe first part of the study suggested that medium-dose Vit C was effective both in preventing the development of NAFLD in HFD-fed mice and reducing HFD-induced liver steatosis,especially in preventing NAFLD development.Based on the above results,we further investigated the effects of medium-dose Vit C,ursodeoxycholic acid(UDCA),and their combination on the development of NAFLD in HFD-fed mice.MethodsC57BL/6J mice were randomly divided into five groups(six mice per group): control group,high-fat diet group,medium-dose Vit C group(30 mg/kg per day),UDCA group(30 mg/kg per day),medium-dose Vit C+UDCA group.All mice received either a normal chow,an HFD,an HFD supplemented with medium-dose Vit C,an HFD supplemented with UDCA,or an HFD supplemented with medium-dose Vit C and UDCA for 15 weeks.Body mass,liver wet weight,liver index,perirenal adipose tissue mass weight,HOMA-IR,serum index(ALT,AST,TC,TG,Glu)levels,and HE and Oil Red O staining histopathological analysis were observed.ResultsCompared with control group mice,mice in high-fat group showed significantly increased body weight,liver wet weight,perirenal adipose tissue mass weight,Glu,TG and TC levels,and notable liver steatosis(P < 0.05).The body weight,perirenal adipose tissue mass weight,liver wet weight,Glu and TG levels in the medium-dose Vit C+UDCA group were the lowest,followed by the UDCA group,which were significantly lower than those in high-fat group(P<0.05).Mice in medium-dose Vit C group showed significantly decreased body weight and perirenal adipose tissue mass weight compared with high-fat group(P < 0.05),Glu and TG levels were reduced in medium-dose Vit C group,but no significant difference were observed(P > 0.05).Mice in high-fat group exhibited prominent liver steatosis,no liver steatosis was observed in medium-dose Vit C+UDCA group,slight liver steatosis was observed in UDCA group and medium-dose Vit C group.And the scores of liver steatosis in the three groups were lower than those in high-fat group,significant difference were observed in UDCA group and medium-dose Vit C+UDCA group(P < 0.05).ConclusionsBoth medium-dose Vit C and UDCA can slow the development of NAFLD to some extent,and the co-administration had a synergistic effect.