| OBJECTIVE:Based on the results of previous research on Wuwu Dan, the clinical safety evaluation model were formed scientifically with evaluation the toxicity on rat and exploration its apoptosis mechanisms.METHODS:The Wuwu Dan induced apoptosis on rat kidney and protein expressions of Bax/Bcl-2as well as NF-κB were determined with TUNEL, immunohistochemical methods and Western Blot analysis, respectively.RESULTS:The renal coefficients were enhanced significantly at day7,day21and day28in the experimental groups of high, middle and low dosage Wuwu Dan with p value<0.05or<0.01. The thymus coefficients of high dosage group were decreased significantly at day7, day28as well as increased level of AST and ALT at day14and BuN at day28with p value<0.01.The pathological damages of rat renal with Wuwu Dan external application were the enlargement of renal tubular with degeneration and necrosis accompanied without significant damage of glomeruli, with peak produced at1to2weeks after application of high dosage Wuwu Dan and the peak value decreased along with the duration of application till the4weeks of the lowest damage with regeneration of renal tubular. In addition, there were toxicities on utero-ovarian system and inhibition effects on the sperm generation of testis. The cells of thymus cortex part became necrosis at1week and atrophy to light to middle degree at2to4weeks after application, followed by recover to its normal form. The apoptosis effects induced by Wuwu Dan produced strongest at1to2weeks and weakened after extended application to the lowest damage at1week after application withdrawal.The protein expressions of Bax/Bcl-2, whose peak time was from1week to2week after application with p value<0.05or0.01, followed by reduction of its toxic effects along with extended application.The protein expressions of NF-κB increased significantly from1week to2week after application with p value<0.01and stable at a high level to3to4weeks, followed by level decrease till the date of5weeks.Conclusions:1. The morphological damage of Wuwu Dan to rat organs focused on renal tubular with glomeruli intact. The hepatic morphology damaged insignificantly, though the function were abnormal. There were different degree damages on utero-ovarian, testis and thymus.2.There were dosage positive effects of Wuwu Dan on the renal toxicity, which means the higher dosage, the more serious damage to renal. High dosage of Wuwu Dan got to its peak damage at1to2weeks and middle to low dosage at2weeks, followed by reduction of its damage at1week after application withdrawal, which showed that the damage of the dosage and duration under this research was reversible.3.There were dosage positive effects of Wuwu Dan on the renal apoptosis with the peak damage at1to2weeks, followed by reduction of its damage at1week after application withdrawal which reminds us that the apoptosis was reversible under controlled application of dosage and duration.4. There were dosage and time effects of Wuwu Dan on the renal toxicity through the influences on apoptosis induction and the protein expressions of Bax/Bcl-2, whose peak time was from2week to3week after application, which was reversible after withdraw of the dosage. Thus, our body may have some adaptive function of reducing the toxic effects of Wuwu Dan by its ability of metabolism regulation and injury repair.5. Nuclear factor κB (NF-κB) plays key roles in the apoptosis of renal cells induced by Wuwu Dan which may through regulating the relative protein expression of NF-κB. The high toxicity of application with Wuwu Dan, the higher expression of NF-κB, whilst, the longer duration, the lower expression, which means that there may be some degree of adaption in the body to the toxicity of Wuwu Dan with its function of the regulation of metabolism and improving wound healing.6. It can be inferred from the results that the duration of Wuwu Dan application should not extend more than4weeks and the highest amounts of Wuwu Dan application should less than0.15gram per day, which is relative safe for the body for preventing side effects.7. The study of Wuwu Dan toxicity which focused on the apoptosis of tissues may construct a kind of safety evaluating model and produce a novel new research path on the clinical formal application of traditional toxic drugs with safety and effecty. |
