Study On The Mechanism Of Guanxinning Tablets On ZDF Rats With Early Diabetic Nephropathy Based On The "Brain-gut" Axis

Objective To observe the effect of Guanxinning Tablets(GXN)on ZDF rats with early diabetic nephropathy(DN),and to explore its possible protective mechanism from the "brain-gut" axis angle,and provide reference of the clinical application of GXN.Methods 24 male ZDF rats,aged 8-9 weeks,were fed Purina#5008 diet.After 4 weeks,ZDF rats were divided into 4 groups according to their body weight and blood glucose level,and namely model control group(10 mL/kg sterilized water),GXN low dose group(300 mg/kg GXN),high dose group(600 mg/kg GXN)and positive control group(300 mg/kg MET).6 rats in each group,and continuous administration for 8 weeks.Another 6 ZL rats of the same age were taken as the normal control(NC)group(10 mL/kg sterilized water)and fed with a regular diet.The general signs of rats in each group were observed during the experiment.Random blood glucose was measured every 2 weeks,and HbAlc was detected every 4 weeks.After 8 weeks of administration,the 24h urine of rats was collected by metabolic cage method,and the urine ALB content was measured.In addition,the levels of serum BUN,CREA,UA,GSH-PX,SOD,MDA,IL-6,TNF-α,5-HT,ET-1,NO,VEGF,CRF and NE were measured.The animals were sacrificed,and contents of SOD and MDA were determined by brain tissues.The kidney tissues were taken for H&E staining,PAS staining and ultrastructural pathological observation.The contents of rat colon were taken for intestinal flora and short-chain fatty acid analysis,and the contents of intestinal flora metabolites LPS and TB A in serum were measured.Results(1)Compared with NC group,the body weight,random blood glucose,HbAlc,serum BUN,UA,and urinary albumin excretion rate(UAER)of the model control group were significantly increased(P<0.01),while serum SOD and NO levels were markedly decreased(P<0.01).Moreover,serum MDA,IL-6 and TNF-α levels were significantly increased(P<0.01).In addition,renal histopathological observation showed that glomerular hypertrophy,podocyte localized foot process fusion,diffuse expansion of mesangium,diffuse increase of mesangial matrix,renal cystic hyalinosis and tubulointerstitial inflammatory cells Infiltration,the ratio of mesangial area increased/vascular sphere area was significantly increased(P<0.01).Compared with the model control group,blood glucose was significantly decreased at 4 and 8 weeks after GXN treatment(P<0.05).After 8 weeks of administration,serum BUN,24h urinary albumin(U-ALB),UAER and TNF-B in GXN high and low-dose groups were substantially decreased(P<0.01),and GXN high-dose group had significant serum GSH-Px activity.Elevation and IL-6 levels were significantly decreased(P<0.01).Compared with the model control group,random blood glucose was significantly decreased in the 4th week of the GXN low-dose group and at the 4th and 8th week of the high-dose group(P<0.05).In addition,the levels of serum BUN,24h urinary albumin(U-ALB),UAER and TNF-α in the GXN high and low dose groups were markedly decreased(P<0.01).Furthermore,the activity of GSH-PX and IL-6 level of the GXN high-dose group was significantly enhanced and decreased(P<0.01).(2)Compared with the NC group,the activity of SOD and the content of MDA in rat brain tissues of the model control group were significantly decreased and increased after 8 weeks of administration,respectively(P<0.01,P<0.05).Meanwhile,the serum NO level was markedly decreased(P<0.01),while ET-1/NO ratio,5-HT,VEGF and NE levels were significantly increased(P<0.01).Compared with the model control group,serum NO levels in the GXN treatment groups were significantly increased(P<0.01)and the ratio of ET-1/NO were significantly reduced(P<0.01).Moreover,the serum VFGF and NE levels in the GXN high dose group were significantly reduced(P<0.01).The results of intestinal flora analysis showed that the intestinal flora diversity of the model control group was significantly decreased(P<0.05),and the relative abundance of Clostridiales and Erysipelotrichales were significantly decreased(P<0.05).The relative abundance of Acidaminococcaceae,Erysipelotrichaceae and Sutterellaceae were also significantly decreased(P<0.05).Moreover,the Allobaculum.Erysipelotrichaceae-unclassified,Roseburia and Peptococcus abundance were significantly decreased(P<0.05).After treatment with GXN,the intestinal flora diversity of ZDF rats was significantly increased(P<0.05),including significantly increases of Erysipelotrichales.Acidaminococcaceae and Erysipelotrichaceae abundance.Through Heat Map analysis,it was found that Allobaculum,Prevotella,Oscillibacter and Ihubacter may be the target genus of GXN.In addition,the levels of LPS and TBA in the model control group were significantly higher than those of NC group(P<0.01),while the raitos of n-butyric acid and isobutyric acid in the model control group were significantly decreased than those of NC group(P<0.05).Compared with the model control group,the treatment of GXN can markedly reduce the contents of LPS and TBA,and increase of n-butyric acid(P<0.05).Conclusion This study showed that GXN can effectively lower blood glucose,reduce the excretion rate of microalbumin,inhibit the levels of TNF-α and IL-6,reduce the level of oxidative stress,inhibit the thickening of glomerular basement membrane and mesangial hyperplasia,and improve local microvascular permeability,and thus exert anti-DN effect.GXN also can affect the brain-gut axis and intestinal flora pathways,including the regulation of"brain-gut axis" related growth factors and hormones,improve renal hemodynamics,and by improving the composition and structure of the intestinal flora,inhibit the intestinal flora metabolites LPS and TBA,and promote the production of butyric acid,thereby lowering blood glucose and protecting kidney,controlling or delaying the progression of DN.