Research On Effects And Mechanisms Of Yishenhuoxue Formula Granule In Early Diabetic Nephropathy Rats

Yishenhuoxue formula granule?YSHXF granule?which is composed of ginsengradixetrhizoma,radix astragali,fructus schisandrae chinensis and other three medicinal materials is a traditional Chinese herbal compound as clinical prescription.YSHXF granule supplement for diabetic nephropathy?DN?patients has been used in Chinese traditional medicine clinic to help stabilize blood glucose homeostasis,improve renal function and delay DN progression.But the therapeutic effects and precise mechanisms of YSHXF granule are unclear.What's more,the Chinese herbal compound has some problems such as complex chemical compositions,unclear pharmacodynamic substances and metabolic processes in vivo,difficulty in quality standards control.Therefore studies on therapeutic effects and precise mechanisms of YSHXF granule,searching its pharmacodynamic substances may increase its clinical application,enhance its international market to realize the modern development of traditional Chinese medicine and improve new ideas for drugs against DN.Base on network pharmacology and modern pharmacological research of traditional Chinese medicine we took the monomers of monarch drugs in the prescription as objects.Focus on decrease oxidative stress and suppressing TGF-?1/Smads signaling transduction,the searching and text mining of the references were performed.Ginsenoside Rg1?Rg1?and Astragaloside??AS-IV?were chosen as the pharmacodynamic substances of YSHXF granule.The aim of this paper was to investigate the effects and mechanisms of YSHXF granule and its pharmacodynamic constituents Rg1 combined with AS-IV on early DN Rats induced by STZ.1 Effects of YSHXF granule on early DN Rats.Methods:The DN rat models were built by STZ and then randomly allocated into groups:STZ group[10ml/kg/d saline,intragastric administration?ig?],Benazepril group?1mg/kg/d benazepril,ig?,YSHXF low dose group?0.5g/kg/d YSHXF granule,ig?,medium dose group?1.0g/kg/d YSHXF granule,ig?and high dose group?2.0g/kg/d YSHXF granule,ig?.Another rats without STZ were selected as control group?10ml/kg/d saline,ig?.After 8 weeks treatment,the urine samples,plasma samples,kidneys samples were harvested.The levels of?2-MG,mALB and UCr in urine were detected.And levels of BUN,UREA,SCr,NOS,CAT,GSH-PX,MDA and T-AOC in plasma were measured.The left kidneys were analyzed pathological change by H&E and PAS staining,and detected expression of TGF-?1,Smad2/3 and Smad7 by immunohistochemical analysis.The right kidneys were employed to measure the expression of TGF-?1,Smad2/3,Smad7 and CTGF by western blot method.Results:Compared with STZ group,medium and high dose group had protective effects on DN induced by STZ,which were demonstrated as following:1)a significant reduction on levels of KW/BW index?p<0.01?,24hUV?p<0.001?,?2-MG?p<0.001?,mALB?p<0.001?,UAER?p<0.001?,UCr?p<0.001?,SCr?p<0.001?,UREA?p<0.001?and BUN?p<0.001?,while an apparently increment on levels of BW?p<0.001?and CCr?p<0.05?;2)suppression the swell while keeping the normal volume of glomeruli,protecting the mesangial structure,decreasing the glomerular lesion rate and improving the renal pathology;3)an apparent increment levels of NOS?p<0.05?,CAT?p<0.001?,GSH-PX?p<0.001?and T-AOC?p<0.001?,while reduction level of MDA?p<0.001?to decrease oxidative stress;4)a significant inhibition of TGF-?1,Smad2/3 and CTGF overexpression induced by STZ in kidney tissue,while increase Smad7 expression to suppress TGF-?1/Smads signaling activation;5)1.0g/kg was the best therapeutic dose.2 The screening and determination of pharmacodynamic substances in YSHXF granule.Methods:After searching and text mining of the references,Rg1 and AS-?were chosen as the pharmacodynamic substances in YSHXF granule.Then LC-MS method for Rg1 and AS-?measurement was built.The methodological parameters were inspected.After that the contents of Rg1 and AS-?in YSHXF granule were performed.Rats were randomly allocated into groups:medium dose group?1.0g/kg YSHXF granule,ig?,Rg1 group?50mg/kg Rg1,ig?,AS-?group?16mg/kg AS-?,ig?and Rg1+AS-?group?50mg/kg Rg1+16mg/kg AS-?,ig?.The plasma samples were achieved at 0h,5min,15min,0.5h,1h,2h,4h,6h,12h and 24h after taking the medicine.The concentration detection of Rg1 and AS-?were performed and plotted drug-time curve.Results:1)The LC-MS method was satisfactory on methodological parameters for Rg1 and AS-?detection.2)The contents of Rg1 and AS-?in YSHXF granule were 50mg/g and 16mg/g.3)Compared with Rg1 group,the Rg1 plasma concentration in Rg1+AS-?group and medium dose group were apparently increased at 15min²4h after administration?p<0.05?,the same to AUC_0-??p<0.05?,Cmax?p<0.05?and C_24h?p<0.01?.But there were no statistic difference between Rg1+AS-?group and medium dose group.4)Compared with AS-?group,the plasma concentration of AS-?in Rg1+AS-?group and medium dose group were significantly increased at 15min⁶h after administration?p<0.01?,as well as levels of AUC_0-??p<0.05?and Cmax?p<0.05?.But there were no difference for Rg1+AS-?group and medium dose group.3 The effects and mechanisms of Rg1+AS-?on early DN Rats.Methods:The DN rat models were randomly allocated into groups:STZ group?10ml/kg/d saline,ig?,medium dose group?1.0g/kg/d YSHXF granule,ig?,Rg1 group?50mg/kg/d Rg1,ig?,AS-?group?16mg/kg/d AS-?,ig?and Rg1+AS-?group?50mg/kg/d Rg1+16mg/kg/d AS-?,ig?.Another rats without STZ were selected as control group?10ml/kg/d saline,ig?.After 8 weeks treatment,the urine samples,plasma samples,kidneys samples were harvested.The levels of?2-MG and UCr in urine were detected.And the levels of BUN,SCr,CAT,GSH-PX,MDA and T-AOC in plasma were measured.The left kidneys were analyzed the pathological change by H&E staining,and detected expression of TGF-?1,Smad2/3,Smad7 and CTGF by immunohistochemical analysis.The right kidneys were employed to measure the levels of TGF-?1,Smad7 and CTGF mRNA by Real-Time PCR method.Results:Rg1 group,AS-?group and Rg1+AS-?group treatment had a good protective effects on DN compared with STZ group,which was demonstrated as following:1)a significant reduction on levels of 24hUV?p<0.05?,?2-MG?p<0.05?,UCr?p<0.05?,SCr?p<0.05?and BUN?p<0.05?,while an apparently increment on levels of BW?p<0.05?and CCr?p<0.05?;2)keeping the normal volume of glomeruli,protecting the mesangial and glomerular basement membrane structure,decreasing the glomerular lesion rate and improving the renal pathology;3)an apparent increment levels of CAT?p<0.05?,GSH-PX?p<0.05?and T-AOC?p<0.05?,while reduction level of MDA?p<0.05?to increase antioxidant ability;4)a significant inhibition of TGF-?1?p<0.05?,Smad2/3?p<0.05?and CTGF?p<0.05?overexpression,and increase Smad7?p<0.05?expression.And a significantly diminished mRNA transcription of TGF-?1,CTGF but increase Smad7 transcription in kidney tissue to suppress TGF-?1/Smads signaling activation.Compared within Rg1 group,AS-?group and Rg1+AS-?group,we concluded that:1)Rg1 group had better renal protective effects and anti-oxidation than AS-?group;2)AS-?group had better suppression TGF-?1/Smads signaling activation than Rg1 group;3)Rg1+AS-?group had the best renal protective effects,anti-oxidation and suppression TGF-?1/Smads signaling activation within the three groups.Compared between Rg1+AS-?group and medium dose group,we found that the effects of renal protection,anti-oxidation and suppression TGF-?1/Smads signaling activation had no statistic difference between them.Although Rg1+AS-?group had lower effect of Smad7 and CTGF transcription than medium dose group,there were no apparent difference on effect of Smad7 and CTGF translation between them.In conclusion that,YSHXF granule had good therapeutic effects on early DN rats induced by STZ,and its renal protective effects were associated with antioxidant stress and inhibitation TGF-?1/Smads signaling.The contents of Rg1 and AS-?in YSHXF granule were 50mg/g and 16mg/g.For focus on decrease oxidative stress and suppression TGF-?1/Smads signaling transduction,the therapeutic effects of Rg1+AS-?is correspond to YSHXF granule.