The Cytoprotective Protein PTD-FNK Improves LPS-Induced Oxidative Stress Damage Of Boar Testicular Sertoli Cells By Regulating The Keap1-Nrf2 Signaling Pathway

LPS is mainly located in the cell walls of Gram-negative bacteria,and a small amount of LPS in the body can cause a broad inflammatory response.PTD-FNK is a synthetic recombinant protein.It has been found that PTD-FNK inhibits the apoptosis of frozen-thawed boar and buffalo sperm and mice and boar interstitial cells.However,the application of PTD-FNK to the decrease of oxidative stress has not been reported.The objective of this study was to investigate the antioxidant protective effect of PTD-FNK on oxidative stress Sertoli cells(SCs).It provides a theoretical basis for improving the antioxidant capacity and sperm quality of male animals.The specific research contents and results are as follows:Experiment 1,establishment of oxidative stress models of boar SCs.SCs were isolated by two-step enzymatic digestion.Growth status of SCs was observed and their morphology was identified by oil red O specific staining and RT-PCR.LPS was a damage inducer and the extent of its damage to SCs was measured by CCK8 and MDA and SOD kits.SCs were affixed after 4 h culture.After 12 h,the SCs were almost fully attached and had a clear overall profile.After 24 h culture,the SCs extended their tentacles in an irregular pattern and began to proliferate.After 48 h culture,SCs gradually formed monolayer adherent cells.The cell stained by Oil red O matched SCs.RT-PCR results showed that the cells expressed SCs markers,Sox9 and GATA4.Oxidative stress damage was more likely induced at 12 h of LPS induction at 100 mg/L(P < 0.05),which caused significantly increased MDA and downregulated SOD(P < 0.05).In brief,the oxidative stress model was successfully established.Experiment 2,protective effect of PTD-FNK on LPS-induced oxidative stress SCs.The optimal concentration and time of PTD-FNK protection against SCs were firstly screened by CCK8.The trial was then divided into three groups(Control,LPS,LPS+PTD-FNK).Logarithmic SCs with good growth status were selected.The LPS SCs were induced with 100mg/L LPS for 12 h.The LPS+PTD-FNK SCs were treated protectively with 0.01nmol/L PTD-FNK for 4h before 100mg/L LPS induction for 12 h.The Control SCs were treated with the same volume of DMEM/F12 solution.Trypsin digestion of cells was followed by oxidative stress and ROS kit assays,and total RNA was extracted by Trizol for q RT-PCR.LPS decreased the contents of GSH-Px,T-AOC(P < 0.001)and CAT(P < 0.05)and up-regulated the contents of ROS(P < 0.01)and 8-OHd G(P < 0.05)compared with control group.Compared with control group,LPS down-regulated the m RNA expressions of CAT(P < 0.01),GSH-Px and SOD(P < 0.05).Compared with LPS group,m RNA expressions of CAT(P < 0.01),GSH-Px and SOD(P < 0.001)were up-regulated in LPS+PTD-FNK group.Experiment 3,His pull down technique was used to extract PTD-FNK interacting proteins.The experiment consisted of 2 samples(Con,without recombinant protein;Exp,with recombinant protein).A total of 66 proteins were identified in the Wayne diagram of the differential protein sets between the two groups.The number of unique proteins identified in Con and Exp was 8,41.Identification results showed that vimentin,actin,GRP78,histone and villin-1were the most important.q RT-PCR results showed that PTD-FNK significantly increased the expression of HSPA5 and VIM genes GRP78 and vimentin genes(P < 0.01).This was consistent with the results of mass spectrometry analysis.PTD-FNK binds to GRP78 and waveform proteins,improving the structure and function of SCs to resist stress response.Experiment 4,PTD-FNK ameliorated LPS-induced SCs oxidative damage through the Keap1/NRF2 pathway.The trial was divided into five groups(Control,LPS,LPS+PTD-FNK,LPS+ML385,LPS+PTD-FNK+ML385).The first three groups were treated with the same "experiment 2".The LPS+ML385 was treated with 5 μM of ML385 for 2h,then 100 mg / L LPS for12 h.The LPS+PTD-FNK +ML385 was treated with 5 μM of ML385 for 2h,then 0.01 nmol/L PTD-FNK for 4h,and the final 100 mg/L LPS for 12 h.The cells were digested by trypsin and detected according to WB and ELISA instructions.LPS up-regulated Keap1 and down-regulated Nrf2,HO-1 and NQO1 protein and m RNA expressions compared with Control(P < 0.05).Compared with LPS,PTD-FNK significantly down-regulated Keap1 protein and m RNA expression(P < 0.01),and up-regulated Nrf2(P < 0.05),HO-1 and NQO1(P < 0.01)protein and m RNA expression.The addition of ML385 inhibited the antioxidant effect of PTD-FNK.Therefore,it was reversely verified that PTD-FNK protein inhibited oxidative damage of boar SCs through Nrf2 antioxidant pathway.PTD-FNK can interact with GRP78 to regulate endogenous Keap1/Nrf2-HO-1 antioxidant pathway,inhibit the production of MDA,ROS and 8-OHd G,up-regulate the expression of antioxidant factors,and ameliorate LPS-induced SCs oxidative stress injury.