Isoflurane Induced Germ Cells Apoptosis And Its Mechanism In Neonatal Male Rats

ObjectiveThe aim of this study was to investigate the effect on male germ cells induced by clinicalconcentration of isoflurane, and its underlying mechanism. It will provide an implication onisoflurane clinic application, as well as experimental evidence on exploring the mechanism ofreproductive toxicity and protection measure of occupational exposure.MethodsSixty P1d SD male rats were divided into2groups randomly: the control group and theexperimental group. The control group were kept in35℃incubator and exposed to air; theexperimental group were kept in35℃incubator and exposed to1MAC isoflurane+30%O22h,4h,6h respectively. After exposure, sacrificed the rats immediately, then the right testeswere immersed in4%formaldehyde-phosphate buffer solution for TUNEL analysis. WesternBlot and Real-Time PCR were used to analyze the protein and mRNA levels of CleavedCaspase-3/Caspase-3, Bcl-2, Bax and Cytochrome C.ResultsTUNEL positive cells were detected in all experimental groups. Significant increase of theTUNEL positive cells per tubule were observed in isoflurane2h,4h and6h exposure. Asexpected, all the experimental groups showed a conspicuous decrease in the expression levelof Bcl-2protein, and the expression level of6h exposure group was lower than4h exposuregroup (P<0.05).The expression of Bcl-2mRNA was significantly down-regulated in the4hand6h experimental groups (P<0.05). In addition, its expression level of2h,4h,6h exposuregroup were declined in order (P<0.05). Compared with control group, the increased Bax protein expression was observed in3experimental groups (P<0.05), and its expression in4hexposure group rose slightly than the2h exposure group (P<0.05). The expression tendencyof Bax mRNA, in contrast with the control group, was up-regulated in4h,6h exposure group(P<0.05). The expression level of Bax mRNA in2h,4h,6h exposure group was up-regulatedin order (P<0.05). Compared with control group, all experimental groups showed aconspicuous increase in the expression level of Cytochrome C protein (P<0.05), moreover,4h,6h experimental group showed up-regulated expression tendency of Cytochrome C mRNA(P<0.05). Compared with control group, there was an obvious up-regulation in the expressionof Cleaved Caspase-3protein and Caspase-3mRNA in3experimental groups (P<0.05).ConclusionThe study revealed that exposing to1MAC isoflurane2h could cause apoptosis of P1d rattestis germ cells. After treated with isoflurane6h, time-dependent manner was observed inapoptotic effect. In addition, the results of the apoptosis-related proteins and genes mightprovide us insights into the role of apoptosis in the toxic mechanisms by which isofluraneaffects reproduction in neonatal rat. The Bcl-2/Bax system in mitochondria pathway isinvolved in the apoptosis induced by isoflurane in the neonatal rat germ cells. Nevertheless,further researches are required for having a thorough understanding of the underlyingmolecular mechanism of apoptosis induced by isoflurane.