| Part Ⅰ Active vitamin D delays the occurrence and development of NAFLD by improving the state of oxidative stress.Objective:To explore the effects of active vitamin D on oxidative stress and liver lipid deposition in mice with non-alcoholic fatty liver disease(NAFLD).Methods:1α-hydroxylase[1α(OH)ase]Gene knockout mice was used to construct the model animal of active vitamin D deficiency(KO mice),and the NAFLD mouse model was established by high-fat diet for 10 weeks.The experiment was divided into 8 groups:WT+RD group and KO+RD group with regular diet(RD),WT+HFD group and KO+HFD group of NAFLD model induced by high fat diet(HFD),WT+HFD+NAC group and KO+HFD+NAC group treated with antioxidant NAC drinking water,WT+HFD+VD group and KO+HFD+VD group supplemented with active vitamin D.Serum levels of CHO,TG,AST were examined by biochemical analyzer and Tumor necrosis factor-α(TNF-α)and Interleukin-6(IL-6)levels were measured with enzyme-linked immunosorbent assay(ELISA)kits.The expressions of acetyl-CoA carboxylase(ACC),ATP-citrate lyase(ACL)and fatty acid synthase(FAS)mRNA were detected by qRT-PCR.To assess histological variations,HE section,oil red "O"staining and FAS immunohistochemistry were utilized.The content of malondialdehyde(MDA)was evaluated by TBA colorimetry.We next prepar liver homogenate to detecte the activity of Glutathione peroxidase(GSH-Px)byflow cytometry and mRNA expression of dioxygenase-1 and 2(dual oxidase,DUOX-1/2)by qRT-PCR.Western blot was used to detect the expressions of Oxidative stress related proteins superoxide dismutase-1(superoxide dismutase,SOD-1/2)and peroxidase-1(peroxiredoxin-1,PRDX-1)in liver homogenate.Results:1.Mice were fed RD diet,except that the weight of KO mice is lighter than WT mice.KO mice were lighter than WT mice(KO+RD vs WT+RD,P<0.05),there was no significant difference in liver wet weight and liver index between the two groups.2.NAFLD was induced by HFD diet in mice,the disease indexes of KO+HFD group were significantly higher than those of WT+HFD group:(1)The contents of CHO,TG,ALT,TNF-and IL-6 increased significantly;(2)The mRNA expression of lipid synthesis-related proteases ACC,ACL and FAS were significantly increased as well as the positive rate of FAS immunohistochemical staining(P<0.05);(3)More lipid droplets could be seen in liver oil red "O" staining;(4)We also found that liver wet weight and liver index increased significantly;(5)HE staining showed balloon degeneration and focal necrosis of hepatocytes.It is suggested that active vitamin D deficiency can aggravate the progress of NAFLD in mice.3.The oxidative stress of NAFLD mice induced by HFD diet was significantly increased,and the oxidative stress indexes of KO+HFD group with active vitamin D deficiency were significantly higher than those of WT+HFD group:(1)The levels of liver peroxidation product MDA,pro-oxidation index Duoxl and Duox2 mRNA were significantly increased(P<0.05);(2)The expression of antioxidant index GSH-Px,SOD1,SOD2 and PRDX1 decreased(P<0.05);The results showed that the deficiency of active vitamin D could further promote the oxidative stress response in NAFLD mice.4.NAFLD mice were given antioxidant NAC drinking water or active vitamin D supplementation respectively.The results showed that the levels of MDA,Duoxl mRNA and Duox2 mRNA were significantly decreased in WT+HFD+NAC group and KO+HFD+VD group,while the expression of GSH-Px,SOD1,SOD2 and PRDX1 were significantly increased,and lipid droplet deposition,hepatocyte balloon degeneration and focal necrosis in liver were significantly decreased.Conclusion:Active vitamin D supplementation can alleviate the course of NAFLD by improving the level of oxidative stress and reducing steatosis in the liver of mice.Part Ⅱ Active vitaminD improves the development of NAFLD by inhibiting the p53-p21 signaling pathwayObjective:To explore the mechanism of how active vitamin D improves the progression of NAFLD in mice through antioxidant stress.Methods:The experimental animals were divided into 8 groups:WT+RD group and KO+RD group with regular diet(RD),WT+HFD group and KO+HFD group of NAFLD model induced by high fat diet(HFD),WT+HFD+NAC group and KO+HFD+NAC group treated with antioxidant NAC drinking water,WT+HFD+VD group and KO+HFD+VD group supplemented with active vitamin D.TUNEL and Senescence-associatedβ-galactosidase(SA-β-Gal)staining were conducted to assess the senescence and apoptosis of hepatocytes.Western blot was used to detect the expression of ACC involved in lipid synthesis,SREBP1-C,MTTP involved in lipid transport and signal pathway molecules p53,p21 and p16.The status of hepatic steatosis was determined by oil red "O" staining and FAS proteins immunohistochemistry.Results:1.Compared with WT mice fed with RD diet,there was no significant difference in the apoptosis rate,the positive rate of SA-(3-Gal staining,the expression of lipid metabolic proteins ACC,SREBP1,MTTP,and the expression of signal pathway molecules p53,p21 and p16 in KO mice(P>0.05).2.NAFLD was induced by HFD diet in mice,the indexes related to liver cell senescence in KO+HFD group was significantly abnormal than that in WT+HFD group:(1)The apoptosis rate of hepatocytes,the positive rate of SA-β-Gal staining and the expression of lipid synthesis proteins(ACC,SREBP1)were significantly higher(p<0.05);(2)The expression of lipid transporter MTTP decreased(p<0.05);The results showed that active vitamin D deficiency could promote the senescence of liver cells and lipid deposition in NAFLD mice.3.Antioxidant NAC drinking water or active vitamin D supplementation could significantly reduce the expression of p53,p21,p16 and lipid synthesis proteins(ACC,SREBP1)as well as increase the expression of MTTP protein.3.Antioxidant NAC drinking water or active vitamin D supplementation could significantlyreduce the apoptosis rate and the positive rate of SA-β-Gal staining and lipid synthesis proteins(ACC,SREBP1)as well as increase the expression of MTTP protein.4.Compared with the homologous mice fed with RD diet,the expressions of p53,p21 and p16 in liver homogenate of NAFLD mice induced by HFD diet were significantly increased in WT+HFD group and KO+HFD group(P<0.05),especially in KO+HFD group(P<0.05).5.Antioxidant NAC drinking water or active vitamin D supplementation could significantly decrease the expression of p53,p21 and p16.6.The liver p53 gene of WT mice was silenced by lentivirus transfection,and then HFD diet was given to induce NAFLD.Compared with sh-NC group,the expression of p53 and p21 protein in liver of sh-p53 group decreased significantly(P<0.05),oil red "O" staining showed significantly lower steatosis(P<0.01),FAS histochemical staining positive rate and SA-β-Gal positive rate also significantly decreased(P<0.05).Conclusion:Active vitamin D can regulate p53-p21 signal pathway through antioxidant stress,inhibit hepatocyte senescence and apoptosis,reduce hepatic steatosis and alleviate NAFLD.Part Ⅲ Effect of calcitriol on oxidative stress and insulin resistance in patients with nonalcoholic fatty liver diseaseObjective:To investigate the effects of calcitriol supplementation on oxidative stress and insulin resistance in patients with nonalcoholic fatty liver disease(NAFLD).Methods:A total of 91 NAFLD patients were screened and 68 patients were enrolled in this study.According to the level of serum 25(OH)D3,68 patientiiis were divided into three groups:vitamin D deficiencygroup(n=28),vitamin D insufficiency group(n=17)and normal group(n=23).The patients that vitamin D deficiency and insufficiency group received 0.25μg/d of calcitriolfor 12 weeks.In three groups,57 subjects finished the study and were included in the analysis.The levels of the variables were compared before and after supplementation.Results:At baseline,the hsCRP(P=0.043),MDA(P<0.001)in the vitamin D deficient and insufficient group were higher than those in the normal group,on the contrary,SOD(P<0.001)in the 25(OH)D3 deficiency group was lower than those in the normal group,there were statistical differences among the three groups.After adjustingforconfounding factors,hs-CRP,MDA and SOD still exhibit statistically significant differences.In addition,insulin resistance index(HOMA-IR)was also significantly higher than that of vitamin D normal group(P=0.035).After calcitriol intervention,hs-CRP and MDA levels of NAFLD patients in vitamin d deficiency group and deficiency group decreased significantly(P<0.05),SOD activity increased(P<0.05),and fatty liver index(FLI)decreased(P<0.05),but HOMA-IR before and after calcitriol intervention did not change significantly(P>0.05).Conclusion:Calcitriol can improve oxidative stress response in patients with vitamin d deficiency and NAFLD deficiency,but has no obvious effect on insulin resistance. |
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