The Mechanism Of 1,25（OH）₂D₃ In Ameliorating Diabetic Liver Injury By NF-κB Pathway

Objectives1 To establish a diabetic rat model and investigate the effects of 1,25（OH）₂D₃（Vit D）administration（with different dose/different durations）on serum levels of fasting blood glucose,lipid,insulin and transaminase,and hepatic inflammatory cytokines in diabetic rats.2 To explore the molecular mechanism in ameliorating hepatic inflammatory injury in diabetic rats by 1,25（OH）₂D₃.3 To explore the regulatory role of Vit D/VDR signal in inflammatory stress via si-RNA technique.Methods1 Diabetes model establishment and grouping:16 4-week-old SFP,male SD rats were randomly selected as controls（NC group）and fed with normal diet throughout the whole process;the rest 104 rats were fed with high-sucrose and high-fat for 5 weeks,and then injected with streptozocin（STZ）（35 mg/kg）intraperitoneally to establish the diabetic rats model（three times random blood glucose≥16.7 mmol/L at 24 h,72 h,and 1 week were used as the criterion）.Diabetic rats were randomly divided into:model control group（MC group）,high dose Vit D group（H VitD group）,middle dose Vit D group,（M VitD group）and low dose Vit D group（L VitD group）.Rats in NC and MC group were given 2 ml/kg/d corn oil by gavage;rats in H,M and L VitD groups were given 0.3,0.15 and 0.075μg/kg/d 1,25（OH）₂D₃ dissolved in equal volume corn oil,respectively;the intervention period lasted for 4 and 12 weeks.2 The weight of rats were checked weekly;the food,water consumption and urine output of rats for 24 h were measureed at before/after the model establisment,4 and 12 weeks after intervention,respectively.3 After the intervention,the rats were anesthetized to collect blood from the abdominal aorta and then collected liver tissue.Serum FBG,lipid profile（TC,TG,HDL-C and LDL-C）,transaminase levels（AST and ALT）were measured by using automatic biochemical analyzer.Serum 25（OH）D,insulin and hepatic homogenized inflammatory factors（TNF-α,IL-1β and IL-6）concentrations were analyzed by using ELISA kits.HE staining was uesed to observe the hepatic pathological changes.Trizol method was used to extract total RNA of liver tissue,then qPCR was used to detect the relative mRNA expression of Vdr and NF-κB signaling molecules（Ikkβ,Iκbα and p65）.Western blot（WB）was used to determine the protein expressions of VDR,IKKβ,IκBα,p-IκBα,and p65 of liver tissue.The localization of VDR and IκBα in hepatocytes were detected by immunofluorescence.4 LO2 cells were stimulated with HGPA（33.3 mmol/L high glucose+0.1 mmol/L palmitic acids）for 24 h to establish an in vitro inflammatory model,and then were divided into control group,model group（HGPA）and intervention group（HGPA+Vit D）.The levels of TNF-α,IL-1β and IL-6 in LO2 cells were detected.5 VDR siRNA was transfected into LO2 cells with GP-siRNA-Mate plus.The mRNA and protein expression of inflammatory factors（TNF-α,IL-1β and IL-6）and NF-κB signaling molecules（IKKβ,IκBα,p65）were detected after transfection.6 The proteins of control,HGPA and HGPA+Vit D groups were extracted,and the interaction between VDR and IκBα was detected by co-immunoprecipitation（CoIP）.7 The results of all measurement data conforming to the normal distribution data were expressed by mean ± standard deviation.The comparison between the two groups adopts independent sample t test The comparison between multiple groups adopts one-way ANOVA,and the post analysis was conducted by LSD-t test or Tukey test.P values<0.05 were considered as statistically significant.Results1 A stable diabetic rat model was established by high-sucrose and high-fat diets and combined with low dose STZ,the rats diabetic model share the characteristics of elevated FBG,dyslipidemia,weight loss,polydipsia and polyuria.After 4 and 12 weeks of intervention,rats in H VitD group weighted significantly more than those in MC group（P<0.05）;after 12 weeks of intervention,compared with the MC group,the water consumption and urine output of diabetic rats in all dose Vit D group decreased by 23.8%-31.8%and 18.6%-26.7%（P<0.05）.2 Compared with the MC group,L VitD group after 4 weeks intervention and M and L VitD group after 12 weeks intervention significantly decreased the levels of FGB in diabetic rats（P<0.05）;the levels of insulin were significantly increased in all dose Vit D groups after 12 weeks intervention（P<0.05）;the levels of TC and TG were significantly reduced after 12 weeks intervention in the H VitD group（P<0.05）,and the levels of HDL-C and 25（OH）D were significantly increased in all dose Vit D groups after 4 and 12 weeks intervention（P<0.05）;but the levels of LDL-C were not significantly affected by Vit D intervention.3 Compared with the MC group,after 4 weeks intervention,the levels of AST and ALT in all dose Vit D groups decreased by 20.9%-46.9%and 17.1%-19.2%（P<0.05）;the levels of TNF-α,IL-1β,and IL-6 in liver tissues of all dose Vit D groups had a significant decrease after 12 weeks intervention（P<0.05）.4 The results of HE staining showed that 4 and 12 weeks of Vit D intervention significantly decreased the pathological scores of liver tissue in all dose Vit D groups（P<0.05）.The results of immunofluorescence showed that VDR was mainly distributed in nucleus,and IκBα was mainly distributed the cytoplasm;after 12 weeks of intervention,IκBα in H VitD group moved more closer to the nucleus,while merging into the nucleus for VDR binding.5 Compared with the MC group,the levels of VDR mRNA and protein expression in H and M Vit D groups were significantly increased after 4 and 12 weeks of intervention（P<0.05）;the levels of IκBα and p65 mRNA expressions were significantly up-regulated in all Vit D groups after 4 and 12 weeks of intervention（P<0.05）;the ratios of p-IκBα/IκBα and nuclear p65/cytoplasmic p65 in all Vit D group s decreased significantly after 4 and 12 weeks of intervention（P<0.05）.6 Compared with the Control group,the levels of TNF-α,IL-1β and IL-6 in LO2 cells significantly increased by 84.1%,61.7%,and 82.2%,respectively,after HGPA stimulated for 24 h（P<0.05）.Compared with the HGPA group,the levels of TNF-α,IL-1β and IL-6 significantly decreased by 31.3%,36.8%,and 28.4%,respectively,after Vit D and HGPA co-cultured LO2 cells for 24 h（P<0.05）,suggesting that 1,25（OH）2O3 can inhibit HGPA-induced inflammation activation.7 The results of qPCR and WB showed that siVDR-homo-1414 transfection significantly decreased the mRNA and protein levels of VDR by approximately 74.3%and 72.2%,respectively（P<0.05）.Compared with the wild type HGPA group（WT group）,the levels of inflammatory factors（TNF-α,IL-1β and IL-6）of si-VDR-HGPA group were significantly increased（P<0.05）,suggesting that VDR down-regulation may stimulate the activation of inflammatory response in LO2 cells.8 The results of CoIP showed that no binding of VDR and IκBα was detected after LO2 cells exposure to HGPA for 24 h alone,whereas binding of VDR and IκBα was detected after Vit D and HGPA co-incubation LO2 cells for 24 h,indicating that Vit D may enhance or catalyzes the binding of VDR to IκBα.Conclusion1 1,25（OH）₂D₃ can delay the excessive weight loss,improve the symptoms of polydipsia and polyuria,whlie reducing the FBG levels of diabetic rats,and improving dyslipidemia,but no significant dose and time dependent correlations could be found in all Vit D groups.2 1,25（OH）₂D₃ can reduce the levels of transaminase and liver inflammatory factors in diabetic rats;meanwhile ameliorating hepatopathy injury and inflammatory infiltration.This effect maybe related to the VDR/NF-κB signaling pathway.3 1,25（OH）₂D₃ can inhibit the inflammatory response of LO2 cells induced by HGPA,the protective effect may be related to the mutal binding of VDR and IκBαmediated by Vit D.