The Effect And Mechanism Research Of Ginsenoside Compound K On Diabetic Kidney Disease

Objective: As one of the major risks of end stage renal disease(ESRD),diabetic kidney disease(DKD)lacks effective clinic early intervention,due to its complex pathogenesis.Ginseng is widely used in traditional Chinese medicine for the therapy of DKD in clinic,presenting good efficiency.This study evaluated the efficacy of CK in treating DKD and proceed in-depth mechanism study,aimed to provide scientific basis for ginseng in the treatment of DKD and development of new drug.Methods:(1)The db/db mice were chosen as DKD model and divided into three groups(n=7)according to serum glucose level,including model group(Model),metformin group(200 mg/kg,Metformin)and ginsenoside compound K group(40 mg/kg,Compound K).Wild type db/m mice were used as the normal control(Normal).For the treatment group,db/db mice were supplemented with CK and metformin mixed in the diet for consecutive 16 weeks.Water weight,food weight and body weight of mice were recorded during the experimental period,and blood and urine biochemical indexes of mice in all groups were analyzed regularly.After the final administration,blood samples,24 h urine samples and kidney tissues were collected.Hematoxylin&eosin staining,periodic acid-schiff staining and masson staining methods were used to assess renal pathological changes.Real time quantitative polymerase chain reaction(RT-PCR)analysis was applied to examine renal inflammatory relative m RNA expression such as c-jun N-terminal kinase(JNK),nuclear factor-κB p65(NF-κB p65)and inhibitor of NF-κB-α(IκB-α);fibrotic relative m RNA expression such as plasminogen activator inhibitor-1(PAI-1),type i V collagen(Col4)and vascular cell adhesion molecule-1(VCAM-1);abnormal proliferation relative m RNA expression such as Cyclin A,cyclin-dependent kinases 2(CDK2)and CDK4;and antioxidant relative m RNA expression such as nuclear factor erythroid 2 related factor 2(Nrf2),heme oxygenase-1(HO-1)and NADH quinone oxidoreductase 1(NQO1).Western blot(WB)analysis was used to analyze the expression levels of inflammation and fibrosis related proteins in the TLR4/NF-κB p65/transforming growth factor-β1(TGF-β1)signaling pathway in the kidney tissues.(2)Mice glomerular mesangial cells SV40 MES 13 were treated with high glucose to establish renal injury model in vitro,and then treated with CK at different concentrations for 24 h;and DMEM with low glucose(LG)treatment was used as control.The expression levels of TLR4,NF-κB p65,NOD-like receptor protein 3(NLRP3),TGF-β1 and other proteins were detected by WB.TLR4 si RNA transfected into mesangial cells and TLR4 agonist LPS were separately used to investigate the molecular mechanism.(3)After the final administration of db/db mice,feces were collected and analyzed by 16 sRNA sequencing to assess the effects of CK on intestinal flora of DKD mice;in addition,the serum concentration of imidazole propionate(IMP),a harmful metabolite of intestinal bacteria,was analyzed to investigate the correlation between IMP and DKD.(4)SV40 MES 13 cells treated with IMP were used to set up another renal injury model in vitro and then treated with CK at different concentrations for 24 h.The protein expression levels of NF-κB p65,NLRP3,Caspase-1,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)were detected by WB;and further mechanism research was performed based on TLR4.Results:(1)From the week 12 on,the levels of microalbumin(MALB)and total urinary albumin/creatinine(UACR)ratio in db/db mice were significantly increased,indicating the renal function damaged.After 16 weeks of continuous treatment with CK,the levels of urinary MALB and UACR in db/db mice were significantly reduced.DKD characterized renal pathological injury was also attenuated by CK treatment,including glomerular volume expansion,glomerular inflammatory infiltration and diffuse mesangial matrix amplification.Furthermore,accumulation of glycogen and deposition of collagen fiber in the glomeruli of db/db mice were also improved by CK treatment,indicating renal function was alleviated after CK treatment.Moreover,compared with model group,analysis of m RNA and protein showed CK supplementation can obviously decrease expression levels of inflammatory related JNK,IκB-α,NF-κB p65,IKK-α and IKK-β m RNA,TLR4,TLR2,NLRP3,IL-6 and IL-1β protein,and phosphorylation of IκB-α,mitogen-activated protein kinases(MAPK p38)and NF-κB p65 in kidney tissues of db/db mice.Also CK treatment significantly reduced expression levels of fibrotic related PAI-1 and Col4 m RNA,myeloid differentiation factor 88(My D88),TGF-β1 and PAI-1 protein,and phosphorylation of Stat3 in db/db mice.The results concluded that the protective mechanism of CK against renal injury may be related to the inhibition of TLR4,NF-κB p65 and TGF-β1 signaling pathways against inflammation and fibrosis.(2)In consistent with results in vivo,in HG induced mesangial cell model,CK significantly inhibited the protein expression levels of TLR4,TLR2,NLRP3,Caspase-1,TNF-α,IL-6 and IL-1β,and downregulated the phosphorylation levels of MAPK p38,JNK,IκB-α and NF-κB p65.CK treatment also significantly inhibited the phosphorylation levels of Sma and Mad-related protein 3(Smad3),Stat3 and extracellular regulated kinase 1/2(Erk1/2),and suppressed the protein expression levels of TGF-β1 and My D88.Furthermore,after TLR4 silencing,CK could not reverse the high phosphorylation levels of NF-κB p65 and Stat3 induced by high glucose,and the up-regulation of My D88,IL-6 and TGF-β1 induced by high glucose.However,when SV40 MES 13 cells pretreated with LPS,CK could dose-dependably inhibit LPS-induced high phosphorylation of NF-κB p65 and Stat3,and the high expression of MyD88,NLRP3,IL-6 and TGF-β1.The results showed that CK improved renal injury caused by inflammation and fibrosis mainly depended on inhibiting TLR4.(3)Intestinal bacterial sequencing results showed that the composition and structure of intestinal flora in db/db mice were significantly changed,and the abundance of Firmicutes was decreased while the abundance of Bacteroidetes increased.CK treatment significantly increased the abundance of Firmicutes and decreased the abundance of Bacteroidetes.Further analysis at genus level showed that CK treatment significantly increased the abundance of Lactobacillus and decreased the abundance of Bacteroides.Targeted detection of serum IMP derived from histidine by Bacteroides in db/db mice presented that CK treatment significantly reduced the high level of IMP.Further analysis showed that in db/db mice,IMP level had a significant positive linear correlation with key indicators of renal function such as urinary MALB and UACR;and protein expression levels of TLR4,NF-κB p65 and TGF-β1 in the kidney.(4)SV40 MES 13 cells treated with IMP in vitro exhibited significant increase of NF-κB p65,NLRP3,Caspase-1,IL-6 and IL-1β protein,while the abnormal high protein expression induced by IMP was significantly down-regulated by CK treatment.With TLR4 silenced,CK treatment could not recover IMP-induced abnormal phosphorylation of NF-κB p65 and Stat3,and increased protein expression of My D88,IL-6 and TGF-β1.In addition,with LPS pretreatment,CK significantly inhibited the LPS induced high phosphorylation of NF-κB P65 and Stat3 and the high protein expression of My D88,NLRP3,IL-6 and TGF-β1,both stimulated by IMP.It can be seen that the mechanism of CK treating DKD to reduce inflammation and fibrosis damage is also related to the inhibition of TLR4 signaling pathway triggered by IMP.Conclusion: Based on results both in vivo and in vitro,this study clarified the activity of CK in the therapy of DKD,and basically decleared that the mechanism of CK in improving renal injury of db/db mice was related to regulating intestinal flora and alleviating renal inflammation and fibrosis injury caused by high glucose and harmful bacterial metabolite IMP.It mainly depends on the TLR4-mediated NF-κB p65/TGF-β1 signaling pathway.It further provides experimental basis for ginseng in the treatment of DKD and development of new drug.