| Part One:Reference review:The reseach progress in protecting the injury of kidney podocyte by using Traditional Chinese Medicine.Part Two:Clinical Reseach:Randomized controlled study on the treatment of CKD (1-2 stage) proteinuria with Modified Huangqichifeng DecoctionResearch Background:Chronic kidney disease (CKD) is a general term for various types of renal diseases.In recent years, its incidence has been increasing year by year, which has seriously threatened the life and health of human beings. End stage renal disease (ESRD) is CKD severe and final stage, if not given early active intervention, CKD will soon develop ESRD, which seriously affects the life quality of patients and brins a huge economic burden to family.Proteinuria as the risk factors of CKD progression and an important indicator of clinical curative effect, given active intervention in the early stage of CKD is essential to delay the progression of CKD.Modified Huangqichifeng Decoction (MHD) is a commonly used prescription to treat CKD of proteinuria by professor Zhang Yu. The early single case randomized controlled studies have confirmed that it can reduce proteinuria, this experiment through randomized controlled further study in reducing proteinuria.The data of this paper is part of the project of the basic scientific research business expenses of the Chinese Academy of Traditional Chinese Medicine.Objective:To observe the effectiveness and safety of MHD in the treatment of CKD (l-2stage) proteinuria.Methods:In strict inclusion and exclusion criteria, the patients were divided into treatment group and control group.The treatment group was treated with MHD and basic treatment,and the control group was given Benner Pury and basic treatment.Every 4 weeks record Traditional Chinese Medicine(TCM) symptoms integral and 24 hour urinary protein, respectively in 0 week、12 weeks、24 weeks were detected routine blood, liver and kidney function, continuous observation 24 weeks.Resultes:①Comparison of TCM symptoms integral:After 12 weeks and 24 weeks treatment, the TCM symptoms integral of treatment group were significantly decreased (P<0.01),but the control group was not significantly decreased (P>0.05).After 12 weeks and 24 weeks in the treatment group, compared with the control group, TCM symptoms integral decreased significantly (P<0.01).(2)Comparison of 24 hours urine protein:After 12 weeks of treatment, the two groups were not significantly decreased compared with before treatment(P>0.05).After 24 weeks, the two groups were significantly decreased than before treatment(P<0.01).After 12 weeks and 24 weeks, the two groups were compared, the difference was not statistically significant(P>0.05).@Effects on HGB, ALT, AST and Scr:The treatment group in 0,12,24 weeks HGB was higher than the control group(P<0.01); in 12 weeks and 24 weeks after treatment, the Scr level was lower than that before(P<0.01). There was no difference between two groups of ALT and AST before and after treatment(P>0.05).Conclusion:MHD can improve TCM symptoms, and reduce the 24 hour urinary protein.During the observation period hemoglobin was not decrease, liver and kidney function was not damage.so MHD in the treatment of CKD (1-2stage) proteinuria is effective and safe.Part Three:Experimental Reseach Experiment One:Effect of MHD on podocyte injury related proteins like nephrin, podocin, desmin expression by adriamycin inducedResearch Background:Podocyte is one of the inherely glomerular cells, with glomerulus endothelial cells,basement membrane composed of the glomerular filtration membrane.Podocyte as the final barrier of glomerular filtration membrane plays an important role to prevent albumin and other macromolecules as well as negatively charged material leakage. In recent years, with the deepening of research on the podocyte,podocyte injury related protein research have also made great progress, the podocyte has a lot of protein,which is closely related to its structure and function, such as the podocyte slit pore membrane protein-nephrin、podocin and podocyte cytoskeleton-desmin, these protein on maintaining the podocyte structure and function is very essential.Objective:To observe the protective effect of MHD on adriamycin induced podocyte injury.Methods:This experiment using in vitro culture podocytes, induced by adriamycin (ADR) produced type of podocyte injury and with MHD containing serum intervening, CCK-8 to detect change of podocyte viability in different groups of serum containing. Western-Blot was used to detect the expression of podocin、nephrin and desmin in the related protein of podocyte injury in order to reveal the protective effect and mechanism of Modified Huangqichifeng Decoction on the injury of podocyte.Resultes:①CCK-8 showed that compared with the blank group, the podocyte viability of the ADR group was significantly decreased (P<0.01);high and middle group of TCM and Telmisartan group significantly increased the viability of podocyte compared with model group(P<0.01),there was no significant difference in podocyte viability among the three groups (P>0.05). Comparis on high、middle group and low group of TCM, the podocyte viability was significantly higher (P<0.01).②Western-blot showed that compared with the blank group,podocyte slit diaphragm protein-nephrin、podocin expression was significantly decreased in ADR group, podocyte cytoskeleton protein-desmin expression significantly increased(P<0.01).High、middle group of TCM and Telmisartan group compared to ADR group,nephrin、podocin expression increased significantly, desmin expression was decreased (P<0.01),among the three groups compared there was no significant difference (P>0.05);high、 middle group of TCM compared to low group, nephrin, podocin expression was significantly increased, desmin expression was significantly decreased (P<0.01).Conclusion:MHD can increase the expression of podocyte slit diaphragm protein-nephrin、podocin and decrease the expression of podocyte cytoskeleton protein-desmin,which can protect the function of the podocyte.Experiment Two:The effect of Modified Huangqichifeng Decoction containing serum on excessive autophagy and apoptosis of podocytes induced by adriamycinResearch Background:Adriamycin is widely exists in eukaryotic cells and a process of protein degradation dependenting lysosome,it can remove the damaged organelles and aging of the protein and other macromolecular materials,in the process produced free amino acid and fatty acid,which can again for use by cells in order to maintain the intracellular environment steady state.Almost all eukaryotic cells have the basic level of adriamycin, in contrast, the podocyte have higher levels of adriamycin, which can protect podocyte in a certain extent.Apoptosis in normal circumstances can also maintain podocyte in stability environment.However,adriamycin and apoptosis are double-edged sword. Excessive adriamycin and apoptosis can accelerate podocyte death, thereby undermining the stability of the intracellular environment.Objective:To further reveal protective effect of MHD on ADR induced injury of podocyteMethods:Using in vitro culture podocytes, induced by ADR produced type of podocyte injury, with MHD containing serum intervening, to observe the effect of podocyte of autophagy and apoptosis by adriamycin and the effect of MHD containing serum on podocyte excessive autophagy and apoptosis by ADR inducing.Resultes:①CCK-8 method showed that compared with the blank group, the podocyte viability of the ADR group was significantly decreased (P<0.01).Compared with the ADR group, the podocyte viability of ADR+(3-MA) group was significantly increased (P<0.01).② Transmission electron microscope was used to detect autophagosome:podocyte of somata in blank group is usual, more organelles, clear nucleolus, nuclear membrane smooth edge,and integrity, nuclear chromatin was fine and evenly distributed in the nucleus, no autophagosome by double membrane structure packaged.ADR group compared with blank group,the number of autophagosome increased significantly, volume increased significantly,organelles decreased;in other groups of MHD and Telmisartan containing serum,the quantity and volume of autophagosome reduced more obvious compared to ADR group.③Western-Blot method showed that compared with the blank group, the expression of Beclin-1 and LC3-II was significantly increased in ADR group (P<0.01).High、middle group of TCM and Telmisartan group compared with ADR group, Beclin-1、LC3-II protein expression was significantly decreased (P<0.01), there was no significant difference between the three groups (P>0.05).Beclin-1, LC3-Ⅱ protein expression in high、middle group of TCM was significantly lower than the low group of TCM (P<0.01).④AnnexinV/PI shows that the early apoptosis rate of ADR group was significantly higher than that of the blank group (P<0.01).In other groups of MHD and Telmisartan containing serum compared with ADR group,podocyte early apoptosis rate decreased significantly (P<0.01).Compared high、middle group of TCM with Telmisartan group, P> 0.05, the difference was not statistically significant.Compared with the low group, the early apoptosis rate was significantly decreased in the high and middle group of TCM (P<0.01).⑤Transmission electron microscope was used to detect apoptosome:The blank group of podocyte showed no apoptosome.In ADR group, the organelle reduced and nucleolus of podocytes disappeared, nuclear fragmentation, chromatin condensation and formation of apoptosome.In other groups of Modified Huangqichifeng Decoction and Telmisartan containing serum.The changes of apoptosis of podocyte showed varing degrees of remission.Conclusion:①ADR can induce excessive autophagy and apoptosis of podocyte, which can damage it.②MHD containing serum can protect podocytes by inhibiting excessive autophagy and apoptosis. |
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