Experimental Study On The Therapeutic Effect Of Rhizoma Paridis On Chronic Glomerular Disease

Part 1 Effects and Mechanisms of Rhizoma Paridis on theKidneys of Rats with Membranous NephropathyObjective: Membranous nephropathy (MN) is a common cause of primaryglomerulonephritis in adults and characterized by in situ immune complex deposition.The MN rat models were duplicated by caudal vein administration of cationic bovineserum albumin (C-BSA) to study the reno-protective effects and possible mechanismsof Rhizoma Paridis therapy, which may provide a theoretic basis for their clinicalapplications in the treatment of primary glomerulonephritis.Methods: Thirty seven male SD rats were used in this study. 5 of them wererandomly assigned to the normal control group (NC group).Others(O group) wereused to duplicate the MN model by C-BSA caudal vein administration. After thesuccessful establishment of MN model, twenty six survival rats in O group weredivided into 3 groups randomly as follow: model control group(MC group), RhizomaParidis group(RP group), muitiglycosides tdoterygilwilfordii group(MTW group).Intragastric administrations of corresponding drugs were given to rats in RP group andMTW group respectively while others were given Intragastric administrations ofdistilled water. Twenty four-hour urinary protein excretions at different time points(before experiment, at the end of 3^rd,4^th and 8^th week after the initiation ofexperiment),serum urea nitrogen (BUN),creatinine (Cr),albumin(ALB),triglyceride(TG), and total cholesterol (TC) at the end of 8th week were measured ineach group. Immunofluorescence, light and electron microscopy analysis of renaltissues were performed at the end of experiment. Immunohistochemical assays wereused to detect the expression of collagenâ…£(Colâ…£) and nuclear factor-kappaB p65(NF-κB p65) in glomerulus, and use Reverse transcription polymerase chain reaction(RT-PCR) method to evaluate the expression of fibronectin(FN)mRNA in kidneytissue. Results:â‘ Proteinuria was observed in O group at the end of 3rd week andaggravated at the end of 4th week, while relieved significantly at the end of 8th week inthe 2 treated groups (p＜0.01). Quantitative analysis showed significant differencesbetween 2 treated groups and MC group (p＜0.05).No significance was found between2 treated groups.â‘¡NO significance of serum BUN and Cr levels was found among allgroups. Serum TG and TC concentration increased markedly in MC group whencompared with NC group (p＜0.05).Serum ALB concentration in MC group was lowerthan in NC group (p＜0.05).No significant difference in levels of TG was seen betweenNC group, MC group, and 2 treated groups. TC level was significantly lower intreated groups than that in MC group (p＜0.05). ALB level in treated groups wassignificantly higher than that in MC group (p＜0.05), with no significant differencebetween 2 treated groups.â‘¢Immunofluorecence staining showed granular depositionof IgG and C₃ deposited in the glomerular capillary walls and partial mesangialregions. The fluorescent intensity of IgG and C₃ in treated groups was attenuatedcompared to that in MC group (p＜0.01 or p＜0.05), with no significance among treatedgroups.â‘£Light microscopic observation showed the glomerulus were swellingremarkably, the quantity of cells in glomerulus increased significantly, glomerularbasement membrane (GBM) became thicker and rete pegs formed on GBMsegmentally and plenty of depositions were shown under the epithelia in PASMstaining in MC group. Fewer depositions and thinner GBM in treated groups wereobserved compared to that of MC group with no fete pegs been found. The quantity ofcells, the glomerulus diameter and area of section in MC group was significantlydifferent from that of NC group (p＜0.05), the values of above indexes in treatedgroups were significantly smaller than those in MC group (p＜0.05).⑤Undertransmission electron microscope observation, mass electron-dense matter wasfound to deposit under epithelia, GBM became markedly thicker, the flattened ordisappeared foot processes of podocytes confluenced extensively, and podocytesshowed vacuolar degeneration in MC group. Few subepithelial electron-dense matterwas seen in treated groups, with signs of recovery in some confluenced foot processes.Thickening of GBM in RP and MTW group were not obvious. â‘¥Immunohistochemical assays indicated the levels of Colâ…£and NF-κB p65expression were decreased markedly in treated groups than those in MCgroup(p＜0.01),but the levels of Colâ…£and NF-κB p65 expression were no significantdifference between 2 treated groups.⑦RT-PCR indicated the levels of FN mRNAexpression were decreased markedly in treated groups than those in MC group(p＜0.05).⑧The levels of twenty four-hour urinary protein excretion were correlatedpositively with the levels of Colâ…£(p＜0.05).Conclusions:â‘ The rat model of membranous nephropathy was duplicatedsuccessfully by caudal vein administration of C-BSA.â‘¡Rhizoma Paridis had theeffects of reducing urinary protein excretion, improving hyperlipoidemia andhypoproteinemia. It could inhibit the deposition of immune-complex in glomerulus,reduce the thickening of GBM and facilitate the recovery of pathologic lesions inrenal tissues.â‘¢Rhizoma Paridis can restrain the sediment of extracellularmatrix(ECM) and activation of NF-κB p65 in renal tissue of MN rats, thus amelioratethe damnification of glomerulus and postpone the chronic pathologic progression ofthe kidney, it also indicates Rhizoma Paridis inhibit inflammation reaction byregulation activation of NF-κB in renal tissue of MN rats.â‘£The efficacy of RhizomaParidis is similar to MTW. Part 2 Effects of Rhizoma Paridis Pharmacologic Serumon Rat Mesangial Cell Proliferation, Apoptosisand FN Secretion in vitroObjective: To investigate the effects of Rhizoma Paridispharmacologic serum on rat mesangial cell(MC) proliferation, apoptosis andfibronectin (FN) secretion,and explore the possible mechanism.Methods: By the methods of seropharmacology and MC in vitro culture, the seracontaining drugs of Rhizoma Paridis(low-,moderate-and high-dosages) and thepositive control medicine muitiglycosides trioterygilwilfordii(MTW)andPrednisonum were prepared and have been put together with lipopolysaccharide (LPS)and the in vitro cultured rat MC for 12 hours, 24 hours and 36 hours, the effects of thedrugs-containing sera on the proliferation of MC were observed by Cell CountingKit-8(CCK-8) colorimetric assay respectively;apoptosis and apoptosis rate of MCwere detected respectively by Hoechst33258 fluorescence staining method and flowcytometry;the level of FN in the supematants was measured by enzyme-linkedimmunoabsorbent assay (ELISA). The expression of fibronectin(FN)mRNA and bcl-2mRNA of MC were evaluated by Reverse transcription polymerase chain reaction(RT-PCR) method.Results:â‘ Compared with normal control group, OD humerus of LPS groupssignificantly increased(p＜0.01); Compared with LPS group, OD numerus of allRhizoma Paridis groups of different dosages, MTW group and Prednisonum groupsignificantly decreased(p＜0.01 or p＜0.05). Rhizoma Paridis pharmacologic seruminhibiting MC proliferation presented some certain dosage- dependant and time-dependant effect, among which the inhibitory effects of the high doses of RhizomaParidis were similar to those of MTW at the 12nd hour. Compared with high dosesof Rhizoma Paridis group,the inhibitory effects of MTW was much weakened at the24th and 36th hour; the inhibitory effects of Prednisonum weaken markedly than those of all Rhizoma Paridis groups of different dosages.â‘¡MC apoptosis wasinduced obviously by Rhizoma Paridis pharmacologic serum at the 24th hour,andwhich presented some certain dosage-dependant effect by flow cytometry assay.theapoptosis rates of MTW group were higher significantly than those of all RhizomaParidis groups; Prednisonum pharmacologic serum could not induce MCapoptosis.â‘¢Rhizoma Paridis pharmacologic serum can inhibit FN secretion ofMC,and presented some certain dosage-dependant effect by ELISA,the inhibitoryeffect of high-dosage Rhizoma Paridis pharmacologic serum are stronger than thoseof MTW pharmacologic serum and Prednisonum pharmacologic serum.â‘£TheRT-PCR outcome showed Rhizoma Paridis pharmacologic serum, MTWpharmacologic serum and Prednisonum pharmacologic serum could inhibit FNsecretion of MC,among them there were no significant difference. Rhizoma Paridispharmacologic serum could inhibit expression of MC bcl-2 mRNA, the inhibitoryeffects of Rhizoma Paridis were much weakened than those of MTW pharmacologicserum.Prednisonum pharmacologic serum could not inhibit expression of MC bcl-2mRNA.Conclusion:â‘ Rhizoma Paridis pharmacologic serum can partially inhibit theproliferation of MC induced by LPS and FN secretion of MC. Rhizoma Paridispharmacologic serum inhibiting MC proliferation presented some certaindosage-dependant and time-dependant effect. Restraining effects of Rhizoma Paridispharmacologic serum were superior to those of MTW and Prednisonum.â‘¡RhizomaParidis pharmacologic serum can induce apoptosis of MC,which precipitatedreduction of proliferative MC. Rhizoma Paridis pharmacologic serum can control theexpression of bcl-2 mRNA, which partly elucidate mechanism of Rhizoma Paridispharmacologic serum inducing apoptosis.â‘¢The role of restraining proliferation andpromoting apoptosis was one of the possible mechanisms with which Rhizoma Paridisimproved proliferative glomerulopathy. Part 3 The Effect of Rhizoma Paridis PharmacologicSerum on the Activation of NF-κB in Cultured RatMesangial Cell Induced by LipopolysaccharideObjective: To study effect of Rhizoma Paridis pharmacologic serum on theactivation of nuclear factor-κB(NF-κB) in mesangial cell(MC) induced bylipopolysaccharide (LPS).Methods: By the methods of seropharmaeology and mesangial cell in vitroculture, the sera containing drugs of Rhizoma Paridis(low-,moderate-andhigh-dosages) and the positive control medicine muitiglycosides trioterygilwilfordii(MTW)and Prednisonum were prepared and have been put together with LPS andthe in vitro cultured rat MC for 24 hours,Reverse transcription polymerase chainreaction (RT-PCR) method was adopted to evaluate the expression of MC nuclearfactor-kappaB(NF-κB) mRNA.Results: Compared with normaleontrol group, LPS increased significantly thelevels of MC NF-κB mRNA expression(p＜0.01); Compared with LPS group,Rhizoma Paridis pharmacologic serum, MTW pharmacologic serum and Prednisonumpharmacologic serum decreased significantly the levels of MC NF-κB mRNAexpression(p＜0.01). The role of restraining the expression of MC NF-κB mRNA ofMTW pharmacologic serum was superior to those of Rhizoma Paridis pharmacologicserum,and high-dosage Rhizoma Paridis pharmacologic serum is stronger than thoseof Prednisonum pharmacologic serum.Conclusions: Rhizoma Paridis pharmacologic serum can inhibit the activation ofMC NF-κB induced by LPS.