| BackgroundDiabetic nephropathy(DN) is a common and major chronic microvascular complications of Diabetes Mellitus (DM).The glomerular mesangial cells (GMCs) are mainly affected cells of DN, high blood glucose can activate the extracellular signal-regulated kinase (ERK) pathway of GMCs and increase the expression of prostaglandin E2(PGE2), transforming growth factor-β1(TGF-β1) and fibronectin (FN) and other downstream products. Then, it may cause early glomerular hyperfiltration and increased synthesis of extracellular matrix (ECM) as well as lead to the occurrence and development of DN. At present, the studies on the influence of Chinese herbal compound on ERK pathway in DN are almost vivo animal trials and mostly focus on non-hemodynamic effects, but less vitro experiments were reported. The course of treatment in clinical studies of traditional Chinese medicine (TCM) therapy on DN are short generally, with fewer followed-up for more than2years in domestic. The majority of studies were performed with randomized controlled trial, with single program of medication and therapy as well as low clinical promotion. The evaluation indexes focus on the laboratory indexes and clinical symptoms, lacking of evaluation on endpoint events. Yiqi yangyin huoxue recipe is the empirical recipe for diabetic nephropathy (DN) used by my tutor, reducing urinary albumin excretion rate and improving the quality of life in short-term effect observation. However, its mechanism has not been studied previously and it lacks of long-term efficacy.Objective1.Experimental study rexplore the mechanism of Yiqi yangyin huoxue recipe on diabetic nephropathy from inhibiting mesangial cells multiplication, ERK pathway activation and downstream products expression.2.Clinical researchcmake overall evaluation of long-term efficacy of Yiqi yangyin huoxue recipe on DN, excavate characteristics and advantages of TCM on the disease and provide references to improve the TCM standardized treatment program and the overall efficacy evaluation system.Methods1.Experimental study1.1Object:rat mesangial cells.1.2Interventions:induced mesangial cell proliferation and ERK pathway activation, with high glucose as a stimulating factor, serum containing Yiqi yangyin huoxue recipe and Fosinopril to intervene.Rat mesangial cells were divided into six groups, that is normal glucose group, high glucose group, high glucose+Fosinopril group and high glucose+high, medium, low concentration of traditional Chinese medicine group, based on different cultured conditions.1.3Observation indexes:detected the expression of phospho-ERK1/2(pERK1/2) protein in mesangial cells with Westem-blot assay, the expression of TGF-β1gene with Real time-PCR assay as well as the contents of PGE2and FN in cell supernatant with ELISA assay after6h,12h,24h,48h, and72h culture. Detected mesangial cells multiplication using MTT assay in cultured24h,48h and72h.2.Clinical research2.1Object:patients with DN in our hospital or outpatient department between December2006-December2009.2.2Interventions:with TCM treatment as exposure factors, subjects were divided into integrated traditional Chinese and Western medicine therapy dominating on Yiqi yangyin huoxue recipe (treatment group) and western medicine therapy alone (control group) and were retrospective observed.2.3Observation indexes:determined the efficacy of laboratory parameters, efficacy of clinical symptoms and synthetic efficacy of disease as well as counted the total efficacy of treatment; observed all kinds of ending indexes, such as progress of albuminuria and renal function, ESRD and death as well as counted the incidence of endpoint events; evaluated the quality of life and nutritional status of patients.Results1.Experimental study1.1Mesangial cell multiplications were promoted significantly after24h and48h high-glucose stimulation, but the multiplications trend declined at72h, there was no significant difference compared with control group. Fosinopril group and high concentration of traditional Chinese medicine group could significantly inhibit high glucose-induced mesangial cell multiplications, but no significant differences were observed between groups. Medium concentration of traditional Chinese medicine group could inhibit mesangial cell multiplications at24h, but there was no inhibition in low concentration of traditional Chinese medicine groups.1.2The expression of pERK1/2protein in mesangial cells increased obviously after 6h high-glucose stimulation. The expression increased with time goes and reached a peak at24h, then it became weakened gradually and was at the same levels with the control group at the72h point. Fosinopril group and high concentration of traditional Chinese medicine group significantly inhibited the activation of ERK at6h-48h, there was no significant difference in time points between groups. Medium concentration of traditional Chinese medicine group also showed significant inhibition at6h-24h. However, low concentration of traditional Chinese medicine group had obvious inhibition merely at12h.1.3Compared with control group, the expression of TGF-β1mRNA obviously increased from12h and FN from24h in high glucose group.With time passing, the increasing trend was more significant at72h. High concentration of traditional Chinese medicine group could inhibit the increase of the expression of TGF-β1mRNA and FN at each time point. Fosinopril group had obviously inhibited effect at early stage, while medium concentration of traditional Chinese medicine group had obviously inhibited effect in the latter stage. Low concentration of traditional Chinese medicine group showed no inhibited effect.1.4With the activation of ERK pathway, the expression of PGE2increased significantly at6h and reached the peak at48h in high glucose group. The expression was still obviously increased at72h compared to control group but was decreased compared to the expression at48h. The increase of PGE2was inhibited significantly at12-48h in high concentration of traditional Chinese medicine group and at24h as well as48h in medium concentration of traditional Chinese medicine group. Fosinopril group and low concentration of traditional Chinese medicine group showed no inhibited effect.2.Clinical research105cases were enrolled in the study, including67cases in treatment group and38cases in control group. Follow-up time (from enrolled for the first time to the last follow-up) was2-5years (the median follow-up time is3.6years in treatment group and3.8years in control group).2.1Total efficiency of proteinuria decreasing was52.9%in treatment group and28.6%in control group; there was a significant difference between groups. The overall incidence of proteinuria endpoint events was14.9%in treatment group and34.2%in control group; there was a significant difference between groups. The incidence of early DN developed to clinical stage DN was16.1%in treatment group and47.4%in control group; there was a significant difference between groups. The incidence of urinary protein progress was13.9%in treatment group and21.1%in control group; there was no statistical difference between groups.2.2The total efficacy of renal function was62.5%in treatment group and20%in control group; there was a statistical difference between groups. The incidence of CDK progress was14.9%in treatment group and34.2%in control group; there was a statistical difference between groups. The incidence of Scr progressed from normal to abnormal was3.9%in treatment group and17.9%in control group; there was a statistical difference between groups. The incidence of Scr-value doubled or increased≥50%in patients with abnormal Scr was12.5%in treatment group and50%in control group; there was a statistical difference between groups.2.3The total efficacy of clinical symptoms was58.2%in treatment group and31.6%in control group; there was a apparent statistical difference between groups.2.4The total efficiency of synthetic effect was40.3%in treatment group and21.1%in control group; the difference was statistically significant.2.5The incidence of ESRD was3%in treatment group and13.2%in control group; there was a statistical difference between groups.2.6The mortality is7.5%in treatment group and10.5%in control group; there was no statistical difference between two groups.2.7There is a apparent statistical difference in the universal quality of life and the diabetes-specific quality of life between two groups.2.8The incidence of malnutrition risk was14.9%in treatment group and31.6%in control group; there was a statistical difference between groups.Conclusions1.Experimental study:Yiqi yangyin huoxue recipe could inhibit the multiplication of mesangial cells at early stage, the activation of the ERK pathway and the increased expression of PGE2, TGF-β1, FN and other downstream products; it also had intervention effects on hemodynamic effects and non-hemodynamic effects caused by ERK pathway.2. Clinical research rtraditional Chinese therapy mainly on Yiqi yangyin huoxue recipe could better improve clinical symptoms;delay the progress of early proteinuria; protected the kidneys to slow down the progress of renal function and ESRD proceeding as well as improved the prognosis of kidney in DN; enhanced the quality of life and improved the nutritional status. Innovations1.Experimental study1.1As the empirical recipe for diabetic nephropathy (DN) by my tutor, studied the mechanism of Yiqi yangyin huoxue recipe for the first time.1.2The study observed ERK pathway activation and downstream products expression; studied the mechanism of the drug from hemodynamics and non-hemodynamic two aspects.2.Clinical research2.1Observed the TCM therapy on DN in the long-term efficacy (more than2years).2.2Evaluate the long-term overall efficacy from endpoint events, efficacy of laboratory parameters, efficacy of clinical symptom, synthetic efficacy of disease, quality of life and nutritional state.2.3Used the proteinuria and renal endpoint events as the secondary outcomes, earlier to reflect the differences in efficacy. |
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