Experimental Research For Effect Of Bushenhuoxue Recipe On Hpth (1-34) Intervened Osteoblasts And Long-Term Clinical Observation For Treatment Of Renal Osteodystrophy

Renal osteodystrophy (ROD) has a group of clinical symptoms caused, i.e. mineral and bone metabolism disorders in the body caused by chronic kidney disease and end stage renal disease. It is a common, serious and difficultly treated complication of chronic renal failure (CRF). In almost100%of patients with CRF, ROD occurs at GFR (glomerular filtration rate)<10ml/min.The high transfer ROD is one of the three most common types of bone disease, and is mainly caused by secondary hyperparathyroidism. With excessive PTH, the two functions i.e. absorption and generation in the bone remodeling process is hyperactive, and there is abnormal proliferation in osteoblasts and osteoclasts. For the exchange of information between osteoblasts and osteoclasts, the molecular basis is OPG (expressed by osteoblast) and its ligand RANKL as well as receptor activator of NF-KB (RANK) expressed by osteoclast, that is, OPG/RANKL/RANK system, to which PTH has a regulatory role.At present, for ROD, the western medicine mainly carries out symptomatic treatment, including diet therapy, phosphate binder, active vitamin D analogues, and calcium-sensing receptor agonists&dialysis. These techniques improve clinical symptoms partially in patients with ROD, but clinical long-term efficacy is not ideal in addition to ectopic calcification of soft tissue etc. With the theory of kidney governing bones, Chinese medicine considers a basic pathogenesis with spleen-kidney insufficiency as a root cause and branch excess with phlegm stagnation as a secondary cause, and adopts the method to invigorate kidney and promote blood circulation with self-prepared Bushenhuoxue Recipe. This recipe regulates bone metabolism and improves hyperparathyroidism so therapeutic.1. ObjectivesObserve the effect of Bushen Huoxue-drug serum on proliferation&differentiation and OPG/RANKL gene expression, and explore the molecular mechanism of ROD treatment via the said recipe in the study of hPTH-intervened mouse, in which in vitro simulation of ROD secondary hyperparathyroidism is made. In the follow-up of ROD patients in the group taking said recipe (particular), the assessment for its long-term clinical effect is made from cardiovascular complications, endpoint events, level of bone metabolism, quality of life and nutritional status, so as to provide a relatively objective assessment basis for clinical ROD treatment using Chinese medicine.2. Methods2.1Experimental method①Culture of MC3T3-El-osteoblast, preparing liquid of hPTH(1-34) plus Chinese/Western medicine drug-containing serum, and experimental grouping;②MTT is used to detect hPTH(1-34)-intervened proliferation of MC3T3-E1(respectively cultured in24h,48h, and72h) in Bushenhuoxue recipe group at different concentrations;③ELISA is used to detect hPTH(1-34)-intervened PICP and ALP secretion levels in the supernatant of cell in Bushenhuoxue-containing serum group at different concentrations.④QRT-PCR is used in absolute quantitative analysis for PTH-intervened OPGmRNA/RANKLmRNA expression of mouse osteoblasts at8h,12h and24h.2.2Clinical research methodThe clinical observation objects are the continuous hemodiafiltration patients who were studied in "The clinical study of bushenhuoxue therapy improving the evaluation of quality of life and Bone Metabolism of renal osteodystrophy "form October2008to March2009. According to whether or not to accept Chinese medicine treatment, patients were divided into treatment group and control group. The control group was subject to basic techniques e.g. hemodialysis, regular calcium supplementation. The treatment group in addition to basic techniques was subject to oral Bushenhuoxue particles. Case-control study was conducted. The time of clinical followup observation is3years. There are two clinical Observe Gauge. On one hand, a retrospective record was made in the past3years for number of calcitriol pulse treatments, cardiovascular disease incidence, statistical survival rate, endpoint event rate in the two groups. On the other hand, in end time of Observation,carried out evaluation of quality of life and nutritional status scores in the two groups, obtained laboratory indicators of patients including bone metabolism and renal function the like,and the two groups were compared. Relevant statistical analysis was conducted by SPSS16.0.3. Results3.1Experimental result3.1.1Cell proliferation:Over time, cell proliferation was significantly increased in the Chinese medicine group at different concentrations; model PTH group’s48h proliferation rate began to decline, at72h, model PTH group OD value significantly was lower than the normal group (P<0.05). For the Chinese medicine group at various concentrations, the cell proliferation level was significantly higher than the model PTH group (P<0.01), and this level was dose-dependent.3.1.2Secretion of ALP and PICP:①From48h to72h, ALP secreted in the Chinese medicine-containing serum group at various concentrations was significantly higher than that of the model PTH group (P<0.01);②PICP secreted in the Chinese medicine group at high concentration at72h was significantly higher than the model PTH group (P<0.01).3.1.3Expression of OPGmRNA and RANKLmRNA:①In Bushenhuoxue group at each concentration and western medicine calcitriol group, OPGmRNA level gradually increased over time promoting its expression, and reached a peak in24h; OPGmRNA level increased at each time point compared with the model PTH group. The Chinese medicine group was dose-dependent.②In the Bushenhuoxue group at various concentrations and western medicine group, RANKL expression showed an increasing trend with time, but from12h to24h, RANKL expressed in every group was lower than the model group with a significant difference (P<0.01). In Chinese medicine group at various concentrations, RANKL expression in the high concentration group is lowest, and in the lower concentration group highest.3.2Clinical result3.2.1Calcitriol pulse therapy rate:Compared to the control group, the treatment group could significantly reduce the rate of calcitriol pulse therapies (P<0.05).3.2.2Incidence of cardiovascular complications:the treatment group was significantly lower than the control group in incidence of acute heart failure (P<0.01) and angina frequency (P<0.05).3.2.3Endpoint events:The two groups had a low incidence rate of fractures and showed no significant difference (P>0.05); the two groups had no significant difference in one-year survival rate, but significant in three-year survival rate (P<0.05) in which the treatment were better than the control group. For patients in the two groups, the death was mainly due to cerebrovascular disease and infection.3.2.4Indicators of bone metabolism:Compared to the control group, the treatment group could reduce iPTH, serum phosphate levels and calcium-phosphate product (P<0.05), resulting in a significant difference.3.2.5Indicators of renal function:Between the two groups, there was no significant difference in serum creatinine and blood urea nitrogen levels (P>0.05).3.2.6Quality of life:①Comparison in8aspects:the treatment group was better than the control group in physiological function (PF) and general health (GH), and there was a significant difference (P<0.05); the treatment group had a higher score than the control group in vitality (VT),mental health (MH),function (SF) and emotional role (RE)with a very significant difference (P<0.05).②The treatment group was higher than the control group in total physical health score (PCS) and mental health score (MCS) with a very significant difference (P<0.01)③As to the total score of SF-36, the treatment group was higher than the control group with a significant difference (P <0.05).3.2.7Nutritional condition:12patients (25.5%) had a nutritional risk in the treatment group, while20(47.6%) in the control group; the difference was statistically significant (P<0.05).4. Conclusions4.1Bushenhuoxue recipe can promote MC3T3-E1-osteoblast proliferation, differentiation and maturation under the intervention by hPTH (1-34) in mouse, thereby promotes bone formation improve bone metabolism of osteoblasts under hyperparathyroidism. The promotional effect is concentration dependent.4.2Bushenhuoxue drug-containing serum can up-regulate the expression of OPGmRNA intervention by hPTH (1-34),and down-regulate the expression of RANKLmRNA intervention by hPTH (1-34).indicating that the Bushenhuoxue recipe promote bone formation, suppress excessive bone resorption caused by hyperparathyroidism, improve the balance of bone metabolism.4.3This recipe can reduce the rate of calcitriol pulse therapies, iPTH level, high phosphorus and calcium-phosphate product, improve the quality of life of ROD patients and their nutritional condition, reduce the incidence of cardiovascular complications and improve survival rates. The clinical long-term efficacy is better in the treatment group than the control group.