| BackgroundIn the past 30 years,the prevalence of Diabetes Mellitus?DM?in China has increased17 folders[1],and it has attracted extensive attention.With many complications of diabetes on human health caused a serious threat,among which diabetic nephropathy?DN?is one of the most serious and highest mortality complications in diabetes.DN occurs in type I and type II diabetes,the incidence rate was 15-25%and 30-40%.DN is also the main cause of chronic kidney disease death?CKD?,which is about 50%of the end stage renal disease?ESRD?.From the perspective of Western medicine,the main pathological features of ND are diffuse thickening of glomerular capillary basement membrane,mesangial matrix hyperplasia,and then cause nodular glomerulosclerosis.The main clinical manifestations are proteinuria and special renal morphology and function.Its pathogenesis has not been fully elucidated,but most of the studies show that the occurrence and development of DN is a long-term high glucose multi factors,multi factor based on genetic susceptibility to oxidative stress,lipid metabolism,hemodynamics,vasoactive substances and cytokines and active factor in chronic kidney due to injury,mainly for mesangial cell proliferation and matrix increase,increased collagen secretion,leading to high perfusion,glomerular hyperfiltration,leading to glomerular hypertrophy,glomerular filtration barrier damage,large amount of protein leakage,so that the formation of proteinuria.In the category of traditional Chinese medicine,DN belongs to both thirst and kidney disease.Such as the medical history,diabetes secondary to edema,fullness,turbid urine,and other diseases are closed lattice nephropathy category.DN's pathogenesis of traditional Chinese medicine is kidney deficiency and blood stasis.All kinds of complications of diabetes are yin deficiency and blood stasis.Blood stasis blocking is a characteristic of diabetic nephropathy.Qi deficiency,blood stasis and water wet internal arrest are the basic pathogenesis.While others emphasize the deficiency of Qi and Yin,they also pay more attention to stasis.In short,DN is developed on the basis of diabetes of deficiency of both qi and Yin,long will stasis,long illness must be false,chronic illness and kidney,spleen and kidney two empty,water transport abnormality,caused by adverse gasification.It can be seen that the foothold of DN is in the blood stasis.Through consulting the literature,we found that Chinese medicine Panax notoginseng?NG?,known as“activating blood and stopping bleeding”and“stopping bleeding without leaving stasis”,has a long and extensive history of medication in the treatment of kidney disease by TCM.Our pre experiment also found that Panax notoginseng and its aqueous extract could inhibit the proliferation and collagen secretion of mesangial cells.This combination of TCM and Western Medicine on DN's description of the pathologic features of DN,to inhibit the proliferation of mesangial cells and inhibit the secretion of collagen as the evaluating indicator,screened Dencichine?De?from Panax notoginseng's principal constituents,which can significantly inhibit the proliferation of mesangial cells and inhibit collagen secretion.The mechanism of De to improve the renal injury of diabetic nephropathy was discussed.MethodsFirst,the high glucose-DMEM medium was used to simulate the high glucose environment in diabetes,and the rat glomerular mesangial cells?HBZY-1?were cultured and the DN cell model was created.The inhibiton of the proliferation of mesangial cells and inhibition of the secretion of collagen as the evaluating indicator,through the method of CCK-8 and collagen staining of the 10 effective components of high content in NG was screened out from the De can significantly inhibit the proliferation of mesangial cells and inhibit collagen secretion.MTT,ELISA and immunofluorescence were used to test the inhibitory effect of De on the proliferation of HBZY-1 cells and the inhibition of collagen secretion and accumulation.Secondly,high fat and high sugar diet,combined with intraperitoneal injection of 45mg/kg STZ were used,with the continuous detection of three random fasting blood glucose?FBG?>16.7 mmol/L,and positive urine protein as evaluation index,which indicated the II DN rat model was established.At the same time,the Met,De and NG were orally administered for 9 weeks,the monitoring of the general signs and FBG,at the end of the 9th weeks oral glucose tolerance test?OGTT?was carried out and then anesthesia,biochemical index,insulin level,glycosylated hemoglobin?HbAlc?level and renal function were detected.Finally,rats were sacrificed.The kidneys and pancreas were weighed and fixed or stored at-80?for pathological examination and protein analysis.Thirdly,ELISA,real time-PCR,immunohistochemistry?IHC?,immunefluorescence and Western Blot methods were used to detecte the expression of Collagen I?Col-I?,Collagen IV?Col-IV?,fibronectin?FN?and laminin?LN?,the main components of extracellular matrix?ECM?and the degradation enzyme MMP-9 and TIMP-1 in renal tissue,meanwhile,the protein expression of?-SMA,E-Cad and Vimentin that related to epithelial mesenchymal transition?EMT?were determined.To evaluate the renal protective effect of De from the two mechanisms:reducing ECM accumulation and inhibating EMT.Finally,we used Western blot and Rt-PCR technology to detect the effects of De on the expression of TGF-?/Smad pathway related protein TGF-?1,Smad2/3 and Smad7,and explored possible signal regulatory pathways and their molecular mechanisms.Results1 The effect of main component De in NG on HBZY-1 cell proliferation and itseffect on the accumulation and degradation of total collagen and ECM.De,the main component in NG,significantly inhibited the proliferation of HBZY-1cells at 1×10-3?1×10-4 and 1×10-5 M,and had a better effect than other 9 saponins in NG,and also superior to that of NG ultrafine powder solution.De can also significantly inhibit the proliferation and collagen secretion from HBZY-1 cells induced by high glucose,and its effect was superior to the other 9 saponins,too.Further studies showed that De could reduce the secretion of Col-I,Col-IV,FN and LN induced by high glucose,and increase the ratio of MMP-9 to TIMP-1,and promote the degradation of ECM.2 De correction type DN disorder of glucose and fat metabolism and improvement of renal injury.Compared with the model group,De improved the general signs of DN rats.Biochemical test results showed that De significantly reduced the FBG,insulin,HbAlc levels in DN rats,and improved oral glucose tolerance in DN rats;decreased the triglyceride,total cholesterol,low density lipoprotein and increased high density lipoprotein in serum;protected the kidney function,improved the pathological damage of the kidney and pancreas,reduced the deposition of collagen and glycogen in the kidney tissue,and then prevented the level of renal fibrosis.3 De improvement of ECM deposition in renal tissue and improvement of EMT in renal tissue.De decreased the expression of Col-I,Col-IV,FN and LN,the main components of ECM,in kidney tissue,and significantly increased the ratio of MMP-9 to TIMP-1,to improve the degradation of ECM in renal tissue.In addition,De significantly reduced the protein expression of?-SMA and Vimentin,and also increased that of E-Cad.It is suggested that De can improve renal damage in DN by reducing the accumulation of ECM in renal tissue and inhibiting EMT.4 De alleviated renal injury in DN by inhibiting the TGF-?1/Smad signaling pathway.De significantly reduced the expression of TGF-?1 and Smad2/3,and the endogenous inhibitor Smad7 in TGF-?1/Smad signal pathway,which revealed that the molecular mechanism of De in alleviating renal injury in DN may be by inhibiting the TGF-?1/Smad signaling pathway.Conclusion1 In this study,it was confirmed that NG and De could significantly improve renal injury in DN.NG and De can reduce 24h urine volume and urinary protein excretion,reduce urea nitrogen and creatinine level,reduce collagen,glycogen and ECM accumulation in glomeruli of DN rats,and have significant protective effects on renal dysfunction induced by DN and rational renal damage.2 The function of NG and De can effectively improve the metabolism of sugar and fat in DN rats were found in this study.NG and De can reduce the FBG,insulin level and HbAlc in DN rats,and improve the impaired oral glucose tolerance in DN rats.3 It is confirmed that De is the main ingredient in NG for treating of DN in NG.Both in vivo and in vitro experiments confirmed that De inhibited the proliferation of glomerular mesangial cells,ECM accumulation and collagen secretion,and increased the ratio of MMP-9 to TIMP-1 induced by high glucose,thereby promoting ECM degradation and inhibiting glomerular hypertrophy,and its effect could be compared with that of NG.4 We preliminarily found that De can inhibit the process of renal fibrosis induced by DN.De can reduce the deposition of collagen in renal tissue,decrease the protein expression of?-SMA and Vimentin,and increase the expression of E-Cad,suggesting that it may inhibit the renal fibrosis induced by DN. |
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