Study On Reproductive Toxicity Of Aconitum To Male Rats In Vivo And In Vitro

Aconitum napellus, a kind of Traditional Chinese Medicine widely used in clinic, has many pharmacological actions. In the western medical tradition, Aconitum napellus is used to relieve pain, cardiotonic, anti-inflammatory, anti-tumor and immunolo-regulation. At the same time it is also very toxic. The raw extract of crude Sichuan aconite root, the extract of crude kusnezoff monkshood and salted extract of crude lateral root of aconite belongs to poisonous traditional medicine which are strictly regulated by the SFDA. The main toxins of Aconitum napellus is aconitine. The main target organs are the cardiovascular and central nervous systems. At present, there isn't any report about male reproductive toxicity on aconitum in domestic and foreign literatures.In order to understand male reproductive toxicity on aconitum, we chose three representative aconitum: Saline lateral root of aconite (SLRA), Crude Sichuan aconite root (CSAR), Crude kusnezoff monkshood (CKM), and adopted rat toxicity test in vivo, and sertoli cells and leydig cells culture model to observe their male reproductive toxicity effects.In the rat reproductive toxicity test in vivo, the SD rats were divided into 10 groups: SLRA (1.55,3.09,6.18g raw drug/kg), CSAR (3.3,6.5,13.0g raw drug/kg), CKM (2.1, 4.2, 8.3g raw drug/kg)and negative control using distilled water. The rats were successive administrated for three months, and were observed for one month after drug withdrawal. The results showed that, after administrative period, Compare with negative control group, organ coefficient of testes and epididymis in all dose groups were decreased, and there was a significant difference at the 8.3g raw drug/kg CKM.(P＜0.05). But after convalescent period, there was no significant difference(P＞0.05) in all dose groups compare to negative control. There was no obvious change of histopathology in testes in all dose groups after administrative period and convalescent period.To study the toxic effects of aconitine on rat Sertoli cells in vitro. Sertoli cells were isolated from testis of 18-20 day-old male SD rats. To obtain more Sertoli cells, cultures were hypotonically treated with Tris-Hcl, and identified the Sertoli cells with H.E., Wright-Giemsa staining. Sertoli cells were exposed to one of the following treatments: 0, 5×10~1, 5×10~2, 5×10~3,5×l0~4ng/ml aconitine. Cell viability was determined by the MTT, and concentrations of lactate in media were measured by lactate assay kits. The results showed that, compare with control group(DMSO), the high dose aconitine(5×10~3, 5×10~4ng/ml) induced decrease (P＜0.01) and the low dose aconitine(5×10~2ng/ml)induced light increase in Sertoli cell viability at 48h; aconitine in all dose groups also induced light increase in Sertoli cell viability at 24h. Aconitine stimulated the secretion of lactate from sertoli cells for 24h, there was a significant difference in all dose groups compare to DMSO control group (P＜0.01). The increase rate of secretion amount of lactate descended when aconitine was 5×10~4ng/ml.To study the toxic effects of aconitine on rat Leydig cells in vitro. Leydig cells were isolated from testis of adult male SD rats. To obtain more Leydig cells, testicular cells were centrifuged with discontinuous Percoll gradients, and identified the Leydig cells by 3P-HSD staining. Leydig cells were exposed to one of the following treatments: 0, 5×10~1, 5×10~2, 5×10~3, 5×10~4ng/ml aconitine. Cell viability was determined by the MTT. Contents of MDA and activity of SOD in media were determined by assay kits, and concentrations of testosterone in media were measured by specific radioimmunoassay. The results showed that, cell viability had no significant difference in all dose groups compare to DMSO control group at 24h and 48h (P＞0.05); Contents of MDA and activity of SOD had no significant difference in all dose groups compare to DMSO control group at 24h (P＞0.05)and secretion of hCG-stimulated testosterone had no significant difference in all dose groups compare to DMSO control group at 4h (P＞0.05).Combining the results in vivo and in vitro, we can draw a conclusion: Aconitum napellus(SLRA, CSAR, CKM) in all dose groups have no obvious reproductive toxicity to male rats in vivo. Aconitine in all dose groups have no obvious toxicity on Leydig cell and only the high dose aconitine(5×10~3, 5×10~4ng/ml) has light toxicity on Sertoli cell in vitro.