Protective Effect And Mechanism Of Ginseng Polysaccharides On Diabetic Nephropathy In Db/db Mice

Diabetes,particularly type II diabetes,affects a large proportion of the global population,affecting an estimated 50 billion adults.Diabetic nephropathy(DN)is a common microvascular complication in diabetes.Its course is rapid and irreversible,which seriously threatens the life safety of patients.Therefore,it is of great significance to find drugs to delay DN process and strive for more survival time for patients.Ginseng,the king of all kinds of herbs,contains ginseng saponins,ginseng polysaccharide(GPS),ginseng polypeptides and a small amount of steroids and terpenoids.It was reported that GPS has antioxidant,immunomodulatory,antiinflammatory and other effects.In this study,the protective effect of GPS on DN was observed by db/db mouse model,and its mechanism was preliminarily discussed.Object:To observe the protective effect of GPS on DN in db/db mice and explore its mechanism.Methods:12-week-old db/db mice were randomly divided into Model group,GPS low-dose group,and GPS-high dose group according to blood glucose and body weight,and m/m mice were the Control group.Control group and Model group were given distilled water intragastrically(ig.),while GPS low dose and high dose groups were given GPS 100 and 200 mg/kg ig.once a day for 8 weeks.The body weight and blood glucose of mice were recorded weekly.Urine was collected 1 h after the last administration,and the contents of Urea Nitrogen(BUN),Urine Creatinine(UCR)and Urine Protein(UP)were detected.Blood was collected and serum levels of Total Cholesterol(TC),Triglyceride(TG),High Density Lipoprotein(High Density Lipoprotein,HDL-c),Low Density Lipoprotein(LDL-c),Tumor Necrosis Factor-α(TNF-α),Interleukin-6(interleukin-6,)IL-6)and Interleukin-1β(IL-1β)content were measured.Kidney indexes were obtained from kidney tissues,and Malondialdehyde(MDA)content and Superoxide Dismutase(SOD)activity were measured.Hematoxylin Eosin Staining(HE)was used to observe renal histopathologic changes.The hyperplasia of mesangial matrix and Extracellular Matrixc was detected by Periodic Acid Schiff Stain(PAS),and the degree of renal fibrosis was detected by Masson stain.The thickness of Glomerular Basement Membrane(GBM)was detected by transmission electron microscopy,and the expression level of α-smooth muscle actin(α-SMA)in kidney tissue was detected by immunohistochemistry.The protein expression levels of TGF-β1,p-Smad2/Smad2 and p-Smad3/Smad3 in renal tissues were detected by Western Blot.Results:1.Body weight,blood glucose and kidney index: compared with Control group,body weight and blood glucose in Model group were significantly increased(P<0.01),while kidney index was significantly decreased(P<0.01);Kidney index in GPS 100 and 200 mg/kg groups was Significant improvement(P<0.05),compared with Model group,while there were no significant changes in body weight and blood glucose,both of which were not statistically significant(P>0.05).2.UCR,BUN and UP contents: Compared with Control group,UCR,BUN,and UP in urine of mice in Model group were significantly increased(P<0.01);Compared with the model group,GPS 200 mg/kg could significantly reduce the content of UCR,BUN,and UP in mouse urine(P<0.01),while GPS 100 mg/kg could significantly reduce the content of UP in mouse urine(P<0.05).GPS 100 mg/kg had a trend to reduce the content of UCR,BUN in db/db mouse urine,but there was no statistically significant difference(P>0.05).3.Contents of TC,TG,HDL-c and LDL-c: Compared with Control group,the contents of TC,TG and LDL-c in serum were significantly increased(P<0.01),while the content of HDL-c was significantly decreased(P<0.01)in Model group;Compared with Model group,GPS 200 mg/kg significantly decreased TC content(P<0.01),increased HDL-c content(P<0.05),while GPS 100 and 200 mg/kg significantly decreased the TG and LDL-c contents(P<0.01 or P<0.05).GPS 100 mg/kg could decrease TC content and increase HDL-c content in serum of mice,but there was no statistical significance(P>0.05).4.Contents of TNF-α,IL-1β and IL-6: Compared with the Control group,the serum TNF in the Model group mice-α、 IL-1 β And IL-6 content significantly increased(P<0.01);Compared with Model group,GPS 100 and 200 mg/kg can significantly reduce serum TNF-α And IL-6 content(P<0.01),GPS 200 mg/kg can significantly reduce IL-1 in serum β Content(P<0.01),GPS 100 mg/kg reduced IL-1in serum β The trend of content was not statistically significant(P>0.05).5.MDA content and SOD activity: Compared with Control group,MDA content in Model group was significantly increased(P<0.01),while SOD activity was significantly decreased(P<0.01);Compared with Model group,GPS 100 and 200mg/kg significantly decreased MDA content(P<0.05 or P<0.01),and significantly increased SOD activity(P<0.05 or P<0.01).6.Histopathology:6.1 HE staining: In the Control group,the glomerular structure and morphology of mice were normal and clear,the basement membrane was smooth,the glomerular morphology was regular,the renal interstitium was not abnormal,the renal tubules were neatly arranged,and there was no inflammatory cell infiltration.In Model group,the glomeruli increased in volume,the basement membrane thickened,inflammatory cell infiltration was observed around the glomeruli,and the structure of renal tubules was disturbed and dilated.The histopathology of kidney in GPS 100 mg/kg group was improved.In GPS 200 mg/kg group,the volume of glomeruli decreased significantly,the thickness of basement membrane decreased,only a few inflammatory cells infiltrated,and the renal tubules were closely arranged.6.2 PAS staining: The kidney structure of mice in the Control group was complete and clear,and GBM was smooth,glomerular morphology was regular,renal tubules were arranged tightly and neatly,no inflammatory cell infiltration and glycogen deposition was observed in renal sacs.In Model group,GBM was thickened,ECM was accumulated,and a large amount of purplish red glycogen was deposited in renal microsacs.In GPS 100 mg/kg group,GBM thickened,ECM accumulated,and purplish red glycogen deposition in renal sacs decreased.The glomeruli in GPS 200 mg/kg group were normal,the renal tubules were arranged regularly,the GBM thickness was basically normal,and the renal sacs had a small amount of glycogen deposition.6.3 Masson staining: In the Control group,the renal tubules were arranged normally and the glomerulus morphology was normal,no collagen fiber hyperplasia and tubular basement membrane was observed.In Model group,a large amount of dyed collagen was found in the renal interstitial tissues,and the interstitial fibrous tissues showed bundle and reticular hyperplasia.The glomerular volume of mice in GPS 100mg/kg group was decreased,and the symptoms of fibrous tissue hyperplasia were relieved.The renal tubules in GPS 200 mg/kg group were arranged more regularly and a small amount of collagen fiber hyperplasia was observed.6.4 Transmission electron microscopy: The kidney GBM width of mice in the Control group was normal,and the protrusion was obvious and presented comb tooth shape.In Model group,the protrusion fusion was linear and disordered,ECM hyperplasia and GBM thickening.The GBM thickness of mice in GPS 100 mg/kg group was thinner,the fusion degree of protrusion was reduced,and the hyperplasia degree of ECM was reduced.In GPS 200 mg/kg group,the arrangement of the protrusion was like comb teeth,the fusion of the protrusion was significantly reduced,the thickness of GBM was thinner,and the degree of ECM hyperplasia was reduced.7.Immunohistochemistry: In Control group,α-SMA was not expressed in renal tubules,glomeruli and interstitium,but only in vascular smooth muscle cells.Besides the smooth muscle layer of renal tubules,the α-SMA protein in Model group showed brown-yellow granules in some renal tubule epithelial cells,which were mainly expressed in the GBM side and the cytoplasm of renal interstitial cells.The expression of α-SMA protein in GBM side and renal interstitial cells decreased in GPS 100 and200 mg/kg groups.8.TGF-β1,Smad2/3 protein expression: Compared with Control group,the protein expression levels of TGF-β1,p-Smad2/Smad2,p-Smad3/Smad3 in kidney tissue of Model group were significantly increased(P<0.01);Compared with Model group,the protein expression levels of p-Smad2/Smad2 and p-Smad3/Smad in GPS 100 and 200 mg/kg groups were significantly decreased(P<0.01).TGF-β1 protein expression level in GPS 100 and 200 mg/kg groups was significantly decreased(P<0.05 or P<0.01).Conclusion:GPS can improve kidney function in db/db mice,regulate lipid metabolism,inhibit the release of inflammatory factors,improve antioxidant capacity,reduce kidney damage in DN,and delay the occurrence and development of renal fibrosis.The mechanism of GPS may be related to the inhibition of TGF-β1/Smad2/3 signaling pathway.