Study On Activity Chemical Constituents And Mechanism Of Lespedeza Virgata (Thurb) DC In Glomerulonephritis

Chronic glomerulonephritis(CGN) is a frequently occurring and stubborn disease in the clinic.With the development of CGN,it can be relieved,but can not recovery completely,and died for terminal stage renal failure at last.Therefore,it is necessary to approach the pathogenesis of CGN,to seek the optimal therapeutic regimen and active drug.It can cure earlier period complication of kidney disease, delay the development of it,improve cure quality of patients,and decrease the danger of death.In the experimentation of ischemia-reperfusion for animal kidney,Harris found that the compensated function of alive nephrons was accentuation and its oxygen-consumption increased greatly,so he put forward hypermetabolism theory, which make this researches get a breakthrough in the pathogenesis of nephritis that many etiological factors correlating with glomerulonephritis(GN).For example,the overexpression of cytokine,vasoactive substance and chemotatic factor,the accumulation of extracellar matrix,the proliferation of Mesangial cell and the hypertrophy of renal tubular epithelial cell,are all concerned in an important mechanism-oxidative stress. Lespedeza virgata(Thurb) DC is the principal drug of Shen Yan Si Wei Pian, which is used to cure CGN in the clinic.After analyzing the chemical composition of lespedeza virgata(Thurb) De,we find that it has abundant flavonoids and phenolic acid.And the powerful anti-oxygenation capability of flavonoids and phenolic acid are engaged popular attention.We have described that the flavonoids of lespedeza virgata(Thurb) De has definite therapeutic action for minimal change nephrotic syndrome of rat induced by Adriamycin in our previous experimentations.However, as for ours knowledge,there have been no reports concerning the chemical constituents of this plant are available and whether their phannacodynamic mechanism has relationship with inhibiting oxidative stress.After elucidate the above questions,we can not only find the available compositions for curving CGN, but also further discern the mechanism of protecting kidney from the aspect of anti-oxidation.Mesangial cell,as one of the three inherent cells of renal glomerulus,has active physiologic function,and also play an important role in the occurrence and development process of renal glomerulus inflammation and sclerosis.Previous experimentations indicated that oxygen radicals,as a signaling molecule,gives rise to renal glomerulus sclerosis from activating protein kinase C(PKC) to adjust and control cell proliferation level.In the phase of renal glomerulus diseases,the severity of renal tubule interstitial fibrosis can not be ignored.A great deal of oxygen radicals,which can not be eliminated in time,it can stimulus renal tubular epithelial cell to excrete a mass of extracellular matrix(ECM),for example,fibronectin(FN),laminin(LN) and colloidal matter,which induce renal interstitium fibrosis.In addition,they can also activate nuclear factor KappB(NF-κB),excite the overexpression of fibrosis growth factor,suah as transforming growth factorβ(TGF-β),monocyte chemoattractant protein 1(MCP-1),enlarge the immunological response of kidney, and further damage the interstitial tissue of renal tubule.The topic adopts a chemical activity separation method guided by pharmacology,and uses advanced chromatography technology to isolate active components from lespedeza virgata(Thurb) DC.The structures of these compounds were elucidated on the basis of spectroscopic analysis.At the same time,it applies many experimental medias,such as determine oxygen radical absorbance capacity, cell culture,MTT,immunohistochemistry,Western blotting,flow cytometry and so on,in order to observe the inhibitory action of lespedeza virgata(Thurb) DC to the rat mesangial cell proliferation that caused by lipopolysaccharide(LPS).Analyzed the effect of the active components of lespedeza virgata(Thurb) DC to renal tubular epithelial cell oxidative stress induced by TGF-β₁,discusses the possible mechanism of anti-oxygenation function for renal tubular epithelial cell under pathologic status, and proves its important significance as an antioxidant to prevention and cure GN.Partâ… :Research of lespedeza virgata(Thurb) DC resource.Objective:To know the research progression,pharmacody activity,genera, morphological characteristic and geographical distribution of lespedeza virgata (Thurb) DC.Methods:Summarize the research progression of lespedeza virgata(Thurb) DC by adopting herbal textual and literature investigation method.Results:There are about 26 kinds of lespedeza plant in China,and they are distributed different region.There are flavone,alkaloid,organic acid and other elemental composition in this plant,which have skyscraping medicine value.Conclusions:The resource of lespedeza plant is very exuberant,the price is low, and the pharmacody activity is exact.Therefore,it has good exploitation and utilization value.Partâ…¡Effects of flavonoids from Lespedeza virgata(Thunb) on the proliferation of rat mesangial cells in vitroObjective:To investigate the apoptotic effects of lespedeza flavonoids in rat glomerular mesangial cells(GMCs) and the mechanism of apoptosis.Methods:MTT-based cytotoxicity assay was to test the GMCs proliferation. GMCs were double stained by Heochst33342/PI and observed under fluorescence microscope.AnnexinV/PI staining and flow cytometry were employed for quantitative measurement of apoptosis on GMCs.The content of MDA,activitive of SOD and GSH-PX in GMCs supernatant was measured.Casepase-3 activation was determined by casepase-3 staining Kit.Results:Lespedeza flavonoids significantly inhibited proliferation of GMCs compared with control(P＜0.05).Heochst33342/PI images could discriminate normal cells,apoptosis cells and necrotic cells under fluorescence microscope.The quantitative assay for apoptosis by flow cytometry method,which showed obvious concentration-dependent relationship.The activities of SOD and GSH-PX in 1000 g/ml;200μg/ml;40μg/ml lespedeza flavonoids groups were significantly higher than that in the control group(P＜0.05),as same as concentrations of MDA were significantly lower than that in the control group.Casepase-3 expression was activated by lespedeza flavonoids in a dose-dependent manner.Conclusions:Proliferation inhibition and apoptosis induction of mesangial cells are involved in the mechanisms of the antioxidation effects of lespedeza flavonoids. The latter may be induced by activation of caspase-3. Partâ…¢:Research on chemical active component of lespedeza virgata (Thurb) DC.Objective:To investigate the chemical constituents of lespedeza virgata(Thurb) DC.Methods:Repeated column chromatography and HPLC of the n-BuOH-soluble fraction in the EtOH extract of lespedeza virgata(Thurb) DC.Their structures were elucidated by chemical properties and spectroscopic evideuce.Results:Nineteen components were isolated and identified asβ-rhamno(A1), octadecoic-acid(A2),kaempferol-7-O-L-rhamnopyranoside(A3),7-O--L-rhamnopyra-nsyl -Kaempeferol-3-O-β-D- glucopyranoside(A4),quereetin(A5),E-β-hydroxycinnamic acid(A6),p-rotoeatechuic acid(A7),p-hydroxybenzoic(A8), tricin(A9),kaempferol(A10),lespedezavirgatol(A11),apigenin(A12), lespedezavirgatal(A13),kaempfedtrin(A14),morin(A15),luteolol(A16),quercetin-3-O -α-L-rhamnoside(A17),lespedezacoumestan(A18) and chrysoeriol-7-rutinoside (A19).Conclusions:A11,A13 and A18 are new compounds,A19 was isolated from Lespedeza Michx for the first time,A1,A2,A3,A4,A5,A6,A7,A9,A10,A12,A15 and A16 are isolated from lespedeza virgata(Thurb) DC for the first time.Partâ…£:Research on the anti-oxygenation activity of the three new compositions isolated from lespedeza virgata(Thurb) DC.Objective:To investigate the anti- oxygenation capability of the three new compounds.Methods:We adopt VitaminC as control drug.Determine the anti-oxygenation activity of lespedezavirgatol,Lespedezavirgatal and Lespedezacoumestan by using ORAC method.At the same time,we establish rat kidney injury model inducted by kalitan bromicum(0.7mmol/kg),and determine the capability of eliminating lipid peroxidation by the three new compositions in both plasma and kidney homogenate of renal failure rats.Results:In an oxygen radical absorbance capacity(ORAC),the three new compounds exhibited dose-dependent oxygen radical scavenging activity.All were higher than that of vitamin C.The IC₅₀ values of three new compounds and vitamin C toward malondialdehyd(MDA) levels in plasma and kidney homogenates of kidney failurerats shown the three new compounds could potently counteract oxidative damage on kidney.Conclusions:All of the three new compounds have strong capability to eliminate oxygen radical.Partâ…¤:Research on the function of lespedezavirgatol inhibits the oxidative stress in renal tubular epithelial cell that inducted by TGF-β₁.Objective:To investigate the role of lespedezavirgatol inhibits the reactive oxygen species(ROS) on TGF-β₁ induced accumulation of extracellular matrix (ECM) in human kidney proximal tubular cell-2(HK-2),and discuss its possible mechanisms.Methods:The DCFH-sensitive cellular ROS was measured by flow-cytometry technology(FCM) and confocal microscopy.Immunohistochemical method was employed to detect the expression of FN in intracellular.MNP-9 and NF-κB protein expression were examined by Western blotting.Results:TGF-β₁(8ng/ml) significantly induced the production of intracellular ROS compared with control(P＜0.01).DPI and Lespedezavirgatol can inhibit the TGF-β₁-induced ROS generation(P＜0.01).TGF-β₁ stimulated fibronectin(FN) protein overexpression.Both benazepril and Lespedezavirgatol inhibited the up-regulation of FN(P＜0.05).TGF-β₁ stimulated NF-κB protein overexpression in HK-2 and down-regulated MMP-9 protein-expression,which were ameliorated by lespedezavirgatol and benazepril(P＜0.05).Conclusions:Lespedezavirgatol can inhibit the increasing of ROS level in human kidney proximal tubular cell-2(HK-2) inducted by TGF-β₁.It can inhibit the FN expression and reduce the expression of NF-κB and upregulating the expression of MMP-9,which is likely to be one of mechanisms why lespedezavirgatol can delay glomerular sclerosis and kidney fibrosis.Conclusions(1) There are many anti-oxygenation activity components in the lespedeza virgata(Thurb) DC.(2) Lespedezavirgatol has very strong anti-oxygenation capability,and it can also inhibit the overexpression of FN for the key components in ECM.(3) Lespedezavirgatol can repair the impaired nephridial tissue by decreasing the overexpression of NF-κB and increased MMP-9 protein expression.(4) Lespedezavirgatol is likely to be the active component to curve GN in the lespedeza virgata(Thurb) DC.