| Part 1:Theoretical researchMesangial proliferative glomerulonephritis (MsPGN) turns out to be the most common clinic type in renal glomerular disease, those main pathological features were hyperplasia of glomerular mesangial cell (GMC) as well as more or less increasements of extracellular matrix (ECM) in disease progression. But up till now, there have been no integrate clarification about its pathomechanism. Immunopathogenesis has been validated as a matter of its initial pathogenic factor which has limited direct damage to the kidney, but will activate a series of inflammation-mediated system which will resort severe damage to glomerular and make influences on clinical manifestations. Based on these factors, glucocorticoid and other immunosuppressive agents are now widely used in treating massive proteinuria or any other kind of symptoms, offen brings about side-effects and almost can't inhibit disease progression. As it is well-known that inflammatory cytokines constitute a complicated network and meantime the over-released leads to the so-called "Casada effect", so it is hard to achieve the prospective effects in clinic treatment with single anti-inflamatory cytokines and unpractical with multiple antibodies. Based on the studies about NF-κB who played a central regulatory role in gene expression of inflammatory cytokines, NF-κB inhibitation is now considered to be a new target in anti-inflamatory area. Nowdays, with the further study of Chinese herbs on etiological factor and pathogenesis of MsPGN, people have made prominent advancements and large potential conquering this disease. Chinese pathogenesis for MsPGN is mostly focused on dampness-heat which we believe will acclerate disease progression especially in renal function damage. To dispel dampness-heat is to control the disease, to protect renal function. Ajuga is a local familiar Chinese Herb in fujian province with bittery tasted and cold in nature, which can clear heat and dispel dampness according to TCM theory. As active ingredients, Luteolin has function of anti-inflammatory, inhibiting allergical reaction, anti-proliferation, etc. Based on these TCM theory and current research, we hypothesis that total flavonoids of Ajuga (TFA) has positive treatmental effects on glomerulonephritis. On account of this idea, we worked out the research under the background of no relevant report in Via index or novelty retrieval.Part2:Experimental Research Objective:To observe the effect of total flavonoids of Ajuga (TFA) on Rats with mesangial proliferative glomerulonephritis (MsPGN) and its therapeutic mechanism.Methods:In vivo, we established the improved animal model of chronic serum sickness MsPGN. The model rats with positive urinary protein were randomly divided into the model control group, the tripterygium wilfordii polycoride group (TPG group, 0.018g·kg-1·day-1), the TFA high-dosage group (2.16g·kg-1·day-1), the TFA mid-dosage group (1.08g·kg-1·day-1), and the TFA low-dosage group (0.54g·kg-1·day-1), with the normal rats as control group. After six weeks, 24 h urinary protein, blood biochemistry were detected. Rats Mesangium, GMC and ECM accumulation were observated under the light microscope. The content of MDA and SOD activity was detected by chemical colorimetric assay, the concentration of IL-1, TNF-αby radio immunoassay, the expression of TGF-β1 by ELISA, and the expression of NF-κB p65 in rats nephridial tissue with immunohistochemistry.In vitro, TFA containing serum was prepared, its experimental concentration was determined according to the cell viability and pre-test results. The GMC synchronized in the quiescent culture were divided into following groups:①normal control group (adding 10% normal rat serum),②LPS group (adding LPS 10μg/ml and 10% normal rat serum),③10% TFA groups (adding 10μg/ml LPS and 10% concentration of TFA-containing serum),④5% TFA groups (adding lOμg/ml LPS and 5% concentration of TFA-containing serum),⑤2.5% TFA groups (adding 10μg/ml LPS and 2.5% concentration of TFA-containing serum). After confounding factor added, the cells and medium were collected in 24h and 48h to detect the proliferation of GMC by MTT assay. The expression of FN, Col-IV, and MCP-1 were observated by using ELISA. Mitotic cycle of GMC were detected by flow cytometry in 48h, and RT-PCR was used to detect the expression of MMP-9mRNA, NF-κB mRNA and IκB mRNA.Results:In vivo, the excretion of urine protein in TFA groups and TPG group were more significantly decreased than in model control group (P<0.01 or P<0.05). GMC in model group increased significantly, mesangial region became obviously widen, groundsubstance increased, the capillaries were pressed into narrowing or disappearing, those pathomorphological changed in TFA groups dramatically lessen (P<0.01 or P<0.05). There were no statistics difference in urine protein excretion and pathomorphological change among TFA high-dosage, mid-dosage groups and TWG group (P>0.05). SOD activity in TFA groups were obviously higher than model control group, the expression of MDA, IL-1, TNF-α, TGF-β1 and NF-κB p65 were obviously decreased (P<0.05 or P<0.01), and nuclear translocation of NF-κB was significantly inhibited. The effect of increasing SOD activity and decreasing the content of MDA in TFA mid-dosage group were better than TPG group (P<0.05). The relative analysis shows the expression of NF-κB p65 was significantly positive correlation with the expression of Serum IL-1, TNF-α, TGF-β1 (r=0.566, P<0.05;r=0.669, P<0.05;r=0.598,P<0.05), and was significantly negative correlation with SOD activity (r=-0.825, P＜0.01).In vitro, After 24h and 48h, the cell viability had reached more than 95% in 20%,10%,5% and 2.5% concentration of drug-containing serum group, respectively, with no evident cell toxic action. The proliferation of GMC and the expression of FN, Col-Ⅳ, MCP-1 in TFA-containing serum group at 24h and 48h were obviously lower than those in LPS group (P<0.05 or P<0.01). When acting on GMC for 48h, the percentage of GMC in G1 phases, the expression of MMP-9 mRNA and IκB mRNA in TFA-containing serum group were obviously higher than those in LPS group, while the percentage of GMC in S phases and the expression of IκB mRNA were obviously lower (P<0.05 or P<0.01). The relative analysis showed that the express of NF-κB mRNA was significant negative correlation with the expression of IκB mRNA.Conclusion:The research indicated that TFA had a certain therapeutical effect on MsPGN, including decreasing urine protein excretion; protecting kidney function, inversing the MsPGN pathomorphological change. Its therapeutic mechanism might be related to resist oxidative stress, regulate NF-κB/IκB signal transduction, and successively regulate the expression of cell cycle and inflammatory factors (IL-1,TNF-α, TGF-β1, MMP-9, MCP-1). |
