Study On The Cell Cycle Arrest And Apoptosis Induced By E. Adenophorum In Hepatocytes Of Saanen Goat And Its Apoptosis Pathway

E. adenophorum, a world invasion of noxious weeds, can cause a series of poisoning symptoms and pathological changes of livestock. The study aimed to explore the molecular mechanism of cell cycle arrest and apoptosis induced by E. adenophorum in hepatocytes of Saanen goat. The experiment was composed of 16 healthy Saanen goats which were about 5-6 months and were stochastically divided into control, group Ⅰ, Ⅱ, Ⅲ groups. Goats in each group were respectively fed with 0%,40%,60%,80% E. adenophorumfeedstuff for 3 months. TEM, DNA Ladder, TUNEL, Western Blot, qRT-PCR, FCM and so on were performed to explore the molecular mechanism of cell cycle arrest and hepatotoxicity induced by E. adenophorum. The study was performed to explore the poisonous toxic mechanism and lay the foundation in theory. The results were as follows:1. The different doses of E. adenophorum induced the cell cycle arrest inhepatocytes of Saanen goat. As compared to control, the result of FCM showed that the percentage of hepatocytes in S phase of each experimental groups was significantly decreased (P<0.01). The percentages of Go/G| phase hepatocytes in group Ⅱ,Ⅲ were increased (P<0.05, P<0.01), but group Ⅰ showed no difference (P>0.05). The PI value of each experimental group was decreased with the increased dose of E. adenophorum, and that of group Ⅱ,Ⅲ was significantly decreased(P<0.05, P<0.01), but group I showed no difference (P>0.05). The results showed that E. adenophorum could induce the G0/G1 phase arrest of hepatocytes in Saanen goat.2. The different doses of E. adenophorum induced apoptosis ofhepatocytes in Saanen goat. The results of FCM showed that the normal hepatocytes in experimental groups were significantly decreased as compare to control (P<0.05, P<0.01). Besides, the early and late apoptosis percentage of group Ⅰ, Ⅱ, Ⅲ were significantly increased as compare to control (P<0.05, P<0.01). TUNEL results showed that few apoptotic hepatocytes were observed in control, but many apoptotic hepatocytes stained with FITC were observed in group Ⅱ, Ⅲ.The percentages of apoptotic hepatocytes were significantly increased in group Ⅰ, Ⅱ, Ⅲ(P<0.05, P<0.01) counted from 5 visual fields. The result of AO/EB staining showed that the cell nucleus of hepatocytes in control was uniform and green, and the cell morphology was complete. The cell nuclei of hepatocytes in the group Ⅱ, Ⅲ showed a bright green dense plate, some of which were hyacinth solid shrinkage, and the cell membrane of which was damaged and red. The DAPI staining showed that the nucleus of the hepatocytes is big showing faint fluorescence, but that of group Ⅱ, Ⅲ showed bright fluorescence. The DNA ladder showed that the DNA is a whole band without clear DNA ladder, but the experimental groups showed the whole band in a dose-denpent manner. The TEM results showed that the nucleus of hepatocytes was complete and the distribution of chromatin was uniform. In group Ⅱ, Ⅲ,the apoptotic features of nucleus were chromatin condensation and aggregation.3. The different doses of E. adenophorum activates mitochondria-mediated apoptotic pathway in hepatocytes of Saanen goat. The experiment was conducted through ELISA, Western blot and qRT-PCR to measure the Bax, Bcl-2, Bid, Cytc, APAF-1. PARP, Fas, FasL and Caspase-3,-8,-9 both in protein and mRNA levels, and the caspase-3,-8,-9 activities. The results showed that the chronic poisoning of Saanen goat caused by E. adenophorum induced the decreasing ratio of Bax/Bcl-2, the release of Cyt c from mitochondria to cytoplasm, the formation of APAF-1/Caspase-9 apoptosome and the activation of Caspase-3 and PARP, inducing the dysfunction of mitochondrial. But the Fas. FasL,Caspase-8, Bid protein were not significantly expressed. The above results showed that E. adenophorum induced apoptosis of hepatocytes through mitochondria-mediated apoptotic pathway, but not the extrinsic apoptotic pathway.By feeding Sannen goat on 40%,60% and 80% of E. adenophorum,we can find that E. adenophoruminduced the G0/G1 phase arrest of hepatocytes. Also, the apoptotic morphological detection indicated the apoptosis of hepatocytes caused by E. adenophorum.At last, the mitochondria-mediated apoptotic pathway was trggered by E. adenophorum,but not the extrinsic apoptotic pathway, which was measured by detecting the apoptosis-related factors.