The Study On The Molecular Mechanism Of Gossypol-Induced Apoptosis In Goat Male Germline Stem Cell

Accordding to the 2015 Statistics Bulletin of the National Economy and Social Development published by The China’s National Bureau of Statistics,5.6 million tons cotton were produced in China.The corresponding cotton by-products（cotton seed,cotton seed meal,cotton-seed cake,and cotton stalk,etc）output was also very tremendous.The PRC produced 9.16 million tons cottonseed cake（meal）in 2015,which was accounts for about23%of the world’s total output.If you can solve the toxicity of gossypol,at the current cotton planting,it is enough to meet the protein needs for 500 million people,and it will account for6%of the total consumption of protein.As a widely grown economic crop,cotton is the major oil and protein resource for human and livestock.But the highly toxic of gossypol in cottonseed severely restricts its effective utilization,consequently creating a huge waste of resources.For many years,the related research focused on exploring the cottonseed cake（meal）detoxification methods,but these methods have so many disadventages such as high cost,low efficiency and decline the feed quality.Even more serious problem is it can not solve the the toxic of cotton straw.Above all,these methods greatly hindered the cotton by-products high value-added utilizations.In this case,we should change our mind,studying the mechanism of gossypol toxicity to fundamentally solve the gossypol toxicity.Previous studies have shown the male germline stem cells were the most vulnerable cells in gossypol damages,but the mechanism was still unclear.Therefore,this study focused on the molecular mechanism of gossypol damaged the goat spermatogonium.First in the viability cytotoxic experiments and early apoptosis detected by Annexin V/PI accurately assessed the gossypol toxicity concentration-response relationship.Through variety of probes to study the mitochondria injury and level of reactive oxygen species（ROS）induced by gossypol,we concluded the gossypol damaged mGSCs mechanism in thesubcellular level.Then,the chemical structure foundation of gossypol toxicity was studied by using apogossypolone（ApoG2）,an aldehyde group removed derivative of gossypol.Finally,we applied variety of inhibitors and activators to study the gossypol lesion signaling pathways in mGSCs.The main methods and results of this study in detail are as follows:1.Gossypol obstructed the intracellular ROS elimination,which damaged mitochondria to produce apoptosis.Through the viability cytotoxic experiments and cell cycle analyses,we found gossypol induced cell viability decline due to apoptosis.And the Caspase-9 activity increase in gossypol treatment hinted the mitochondrial apoptosis.Thus,we confirmed the mitochondrial dysfunction by the decreased mitochondrial membrane potential and ATP concentration.The higher intracellular H₂O₂ level did not accompany with the O₂^·-associated increase in gossypol-treated,which indicated that gossypol obstructed the intracellular reactive oxygen species（ROS）elimination.Manipulated gossypol-induced ROS level by H₂O₂ andα-lipoic acid,we found the mitochondrial dysfunction resulted from the excessive intracellular H₂O₂.2.The intramolecular aldehyde group is the key chemical structure of gossypol-induced spermatogonia damage.On the basis of finding out the damage of gossypol from the ROS elimination,the related literatures were extensively searched and the molecular structure of gossypol was analyzed deeply,thus we found that the intramolecular aldehyde group of gossypol may be alkylation addition reaction with the glutathione（GSH）that is the foundation of ROS elimination system.So we treated with Apogossypolone（ApoG2）,an aldehyde group removed derivative of gossypol,as the nagetive control to find out the toxic contribution of aldehyde groups.We found that the GSH/GSSG ratio and H₂O₂ did not decrease in ApoG2group compare with the gossypol group.ApoG2 also did not cause the mitochondrial apoptosis.Hence the aldehyde group is key factor in gossypol cytotoxicity.3.Gossypol induced mitochondrial dysfunction and apoptosis through SIRT1-P53-PUMA pathway.To find the specific signaling pathway of gossypol damage,the levels of mGSCs ROS were manipulated by hydrogen peroxide and lipoic acid,we respectively detected the NAD⁺/NADH ratio,SIRT1 activity.We found that it was due to high ROS levels that increased intracellular NAD⁺/NADH ratio and inhibited downstream SIRT1 activity in gossypol treatment.Western blotting results demonstrated that gossypol increased the P53concentration by inhibiting P53 deacetylation levels,which eventually activated PUMA expression.Comparing with the specific inhibitors groups,the data illustrated that gossypol induced apoptosis through SIRT1-P53-PUMA pathway.In short,we hope to find out some antidotes or feed additives to prevent gossypol toxicity from the guidance of toxicology mechanism in the future.If so,we can fundamentally prevent and cure the gossypol poisoning disease,to serve the key technical problem of the high-quality protein feed lack,which will produce great economic effect.In addition,it can also provide some references for the anti-tumor and male contraceptive application of gossypol derivatives.