Dissection Of Mitophagy Regulated By Atg4B In The Early-weaning Piglets

In animal production, in order to improve the economic benefit, the weaning time of piglets is continuously shortening. But early weaning will force the piglets subject to myrial stressors from physiological and nutrition. Autophagy ubiquitously exists in all eukaryotic cells, which plays an important role in many process of life. Recent studies have shown that the activation of autophagy is necessary for the maintenance of animals survive at the stage of from birth to suck enough milk. However, the physiological role of autophagy induced by the early weaning stress in mammals is still not clear.This project composed of four parts:1. whether the early weaning stress will induces autophagy in piglets.2. Atg4B plays a vital role in the formation of autophagosomes, how the expression of Atg4B influences the activity of autophagy.3. Treatment of HEK293T cells with DMEM-low glucose medium which is not contain serum, Leu, Arg and Lys to simulate the nutrient deprivation caused by early weaning, after2h, utilize Atg4B protein as the bait protein, use tandem affinity purification and mass spectrogram to screen the new proteins that interact with Atg4B in autophagy.Our data showed that:1. The detection of blood physiological and biochemical parameters demonstrate that there had no significant difference(P>0.05) in physiological parameters, but compared with control group(15d), the WBC, RBC, HGB, LY%and MO%are tend to decrease in piglets of weaning24h, and the GR%has a rising trend; In addition, the serum glucose concentration, and the level of insulin and interleukin-6in experimental group(weaning24h) were reduced by31.60%,36.14%,32.11%(P<0.05), glucagon level fell by62.15%(P<0.01), cortisol level increased by67.72%(P<0.05), indicating that weaning at14d lead to nutrient deprivation, the deceased in energy metabolism and immunity.2. Proved by western blot and transmission electron microscopy, after weaning at the time of14d, the level of autophagy in the liver, spleen and piglets of piglets was significantly increased (P<0.01), and within24h after weaning, the activation of autophagy tends to increase.3. Through the immunohistochemical techniques, we found that the expression of Atg4B in the liver or spleen did not change significantly (P>0.05) in14d and15d piglets, which are normal breast-feeding; But24h after weaning, the level of Atg4B was significantly reduced (P<0.01) in the two organizations. 4. The result of tandem affinity purification and mass spectrogram showed that there may be an interaction between Atg4B and LRPPRC, CAND1, IMMT, SLC27A4, or ATP51, suggesting that Atg4B has some kinds of connections with mitochondria in the regulation of autophagy.5. The detection of mitochondrial cytochrome c oxidase subunit COX IV and mitochondrial membrane potential indicated that the starvation stress would impair the mitochondrial oxidative respiratory function, and decrease mitochondrial membrane potential, non-specific inner mitochondrial membrane permeability pore inhibitors Cyclosporin A can inhibit the starvation stress-induced mitochondrial depolarization, and the amplification of autophagolysomes, so mitophagy is taking place in cells to eliminate the damaged mitochondria.In summary, a conclusion came up that early weaning stress can induce autophagy in piglets, and the expression of Atg4B is reduced in the process; In starvation stress, the cells utilize autophagy to maintain intracellular homeostasis and promote cell survival, in addition, initiating mitophagy to remove damaged mitochondria; LRPPRC and other candidate proteins may participate in the regulation of autophagy or mitophagy with Atg4B.