Study On Nephrotoxicity Mechanism Of Cyclovirobuxine D

Cyclovirobuxine D (CVB-D), an active compound extracted from the Chinese medicinal herb Buxus microphylla Sieb. et Zucc. var. sinica Rehd. et Wils, is the active component of huangyangning tablets, which are widely used in China for the treatment of arrhythmias, angina pectoris, and myocardial ischemia. Numerous studies have examined the pharmacodynamic effects and mechanism of CVB-D, but few studies have examined its toxicity. We have found that long-term using CVB-D may cause nephrotoxicology, with the relationship between dose-dependent and time-dependent manners:and the nephrotoxicology cannot be recovered completely after injection. However, few studies have examined with its mechanism of kidney toxicity. In this study, we investigated the mechanism of CVB-D on nephrotoxicity to provide the reasonable reference for clinical practice.1. After CVB-D was ip. given for two weeks, and we tested the relative indicators of urine and serum of rats. We also detected the biomembrane and oxidation indicators of renal cortex and observed pathological changes in renal tissue. Besides, we tested the differential gene expression between the CVB-D20 mg/kg group and control group using gene chip technology. Results showed compared with control group, Glu and TP in urine and BUN, Cr and Cr in serum of CVB-D group was increased; while the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase was significant decreased in CVB-D group. CVB-D had few effects on oxidation indicators of renal cortex. Histopathology showed CVB-D caused capillary ball cell hyperplasia of glomerulus, denaturation and necrosis of renal tubular epithelial cells, and little inflammatory cell in renal interstitial. Gene chip showed CVB-D influenced the pathway about apoptosis, cell cycle, death receptor and so on. Those evidences above suggested that CVB-D caused the nephrotoxicity with ip.for shorter time, and the mechanism was associated with damage of glomerulus and tubles and inhibition of biomembrane activity.2. We observed direct cytotoxic of CVB-D on HK-2cell in vitro by cell viability inhibition, LDH leakage, morphological changes, cell cycle changes and ROS generation. Results showed that CVB-D inhibited HK-2 cells viability and induced LDH leakage at a dose-and time-dependent manner. Treatment of CVB-D increased S phase and the SubGl phase appeared to be simultaneously. In addition, CVB-D had few effects on ROS generation. Those evidences above, especially the appearance of SubGl phase and turned round cells, have suggested that the cytotoxicities of CVB-D may related to apoptosis.3. We observed cell morphology by electron microscope and cell nucleus morphology by Hoechst 33258 staining. Not only did we detect HK-2 cell apoptosis by annexinⅤ/PI staining combined with FCM analysis. We also detected the effects of CVB-D on caspase-3 activity by spectrophotometry and tested the differential gene expression between the CVB-D60μg/ml group and control group using gene chip technology. Results showed CVB-D group HK-2 cell exhibited chromatin margination, which was due to early apoptotic events. AnnexinV/PI staining confirmed the apoptosis was induced by CVB-D in HK-2 cell and without significant necrosis. In the meantime, CVB-D can activated caspase-3 and gene chip results showed it influenced HK-2 cell apoptosis according to caspase cascade, Fas and P53 pathway. All of above showed the CVB-D is able to induce apoptosis on HK-2 cell and caspase-3 is involved.4. We observed the effects of CVB-D on Na+-K+-ATPase activity and tested expression of Na+-K+-ATPaseα1andβ1by western blotting. Besides, we tested the effects of CVB-D on mitochondrial membrane potential and intracellular Ca2+of HK-2 cell. Results showed that CVB-D can significant inhibited Na+-K+-ATPase activity and down regulated expression of Na+-K+-ATPaseα1 andβ1 at a dose-and time-dependent manner. In the meanwhile, CVB-D decreased mitochondrial membrane potential of HK-2 cell and increased intracellular Ca2+. All of above showed a significant decrease in Na+-K+-ATPase activity in cells treated with CVB-D, probably caused by the decrease in the expression of theα1 andβ1 subunit of Na+-K+-ATPase in HK-2 cells that may be associated with the CVB-D-induced apoptosis of HK-2 cells.