Protective Effects Of PTD-FNK On Heat Stress-induced Damage To Boar Sertoli Cells

Sertoli cells（SCs）are one of the important cells that promote the production of normal sperm in the testis,so it is very important to maintain the physiological function of SCs.High temperature can cause heat stress（HS）reaction in boars,which damages the sperm quality of boars and restricts the development of pig industry.PTD-FNK is a synthetic anti-apoptotic protein fused with anti-apoptotic protein FNK and protein transduction domain（PTD）,which has a certain protective effect on cell apoptosis in clinical application.As a novel anti-apoptotic protein,it has not been deeply studied in protecting piglet SCs from HS.Therefore,this study took piglet SCs as the research object,constructed a HS model of boar SCs,and explored the protective effect of PTD-FNK on HS-induced SCs damage,in order to provide a new idea for alleviating the reduction of boar sperm quality caused by high temperature in summer.The specific research contents and results are as follows:1.Establishment of SCs in vitro HS model and screening of PTD-FNK concentration.SCs were screened by CCK8 method under conditions of HS（37℃,39℃,41℃,43℃,47℃）and PTD-FNK（0.01nmol/L,0.1 nmol/L,1 nmol/L,10 nmol/L,100 nmol/L）.The cells were treated with the screened conditions respectively,and the cells were divided into blank group（Control）,heat stress group（HS）,and heat stress+protein group（HS+PR）,and q PCR was used to detect the protective effect of PTD-FNK on the blood-testis barrier of HS-induced damage boar SCs.The viability rate of SCs treated with HS（43℃for 1 h）was significantly decreased（P<0.01）,but the viability rate of SCs pretreated with 0.1 nmol/L PTD-FNK for 1 h before HS was significantly increased（P<0.01）.HS significantly down-regulated the expressions of Claudin-1,ZO-1,Cx43 m RNA（P<0.01）and Occludin m RNA（P<0.05）in SCs.Compared with the HS group,the expressions of Claudin-1,Cx43 m RNA（P<0.001）and Occludin,ZO-1 m RNA（P<0.05）in SCs were significantly up-regulated after PTD-FNK pretreatment.2.Effects of PTD-FNK on apoptosis,antioxidant capacity and mitochondrial function of SCs.QPCR was used to detect the expression of apoptosis genes and mitochondrial dynamics-related genes,and the kits were used to detect T-AOC,SOD activity,MMP,and MDA,ROS,ATP,and Cyt C contents.In terms of apoptosis,compared with the Control group,HS significantly up-regulated the expression of Caspase-3,Caspase-8,Caspase-9,HSP70 m RNA（P<0.01）and Bax m RNA（P<0.001）in SCs,and significantly down-regulated the expression of Bcl-2m RNA（P<0.01）in SCs.Compared with the HS group,HS+PR significantly down-regulated the expression of Caspase-3,Bax m RNA（P<0.01）and Caspase-8,HSP70 m RNA（P<0.05）in SCs,and the expression of Bcl-2 m RNA was significantly up-regulated（P<0.05）.In terms of oxidative stress,compared with the Control group,the MDA content（P<0.05）and ROS content（P<0.001）in the HS group were significantly increased,the T-AOC and SOD activity were extremely significantly decreased（P<0.01）.Compared with the HS group,the MDA content（P<0.05）and ROS content（P<0.01）in the HS+PR group were significantly decreased,the T-AOC（P<0.05）and SOD activity（P<0.01）were significantly increased.In terms of mitochondrial damage,compared with the Control group,the MMP and ATP production in the HS group were significantly decreased（P<0.01）,and the contents of Cyt C（P<0.001）and Ca²⁺（P<0.01）were significantly increased.Compared with the HS group,the MMP（P<0.001）and the ATP production（P<0.01）in the HS+PR group were extremely significantly increased,the Cyt C content（P<0.01）and Ca²⁺content（P<0.05）were significantly decreased.Compared with the Control group,HS significantly up-regulated the expression of Cyt C,Drp1 m RNA（P<0.05）and Opa1 m RNA（P<0.01）in SCs,and significantly down-regulated the expression of Mfn1 m RNA（P<0.01）.Compared with the HS group,HS+PR significantly down-regulated the expression of Cyt C（P<0.01）,Drp1（P<0.001）and Opa1（P<0.05）m RNA in SCs,and the expression of Mfn1 and Mfn2 m RNA were up-regulated（P<0.05）.3.Transcriptomic analysis to the effect of PTD-FNK on anti-heat stress ability of boar SCs.Transcriptome sequencing analysis of the three groups of cells was performed,and q PCR was used to detect whether PTD-FNK could regulate the PI3K/Akt signaling pathway.Compared with the HS group,there were 270 differentially expressed genes（193up-regulated and 77 down-regulated）in the Control group.Compared with the HS+PR group,there were 3 649 differentially expressed genes（1430 up-regulated and 2 219 down-regulated）in the HS group.GO and KEGG pathway analysis of Control group and HS group,HS group and HS+PR group showed that the differentially expressed genes were enriched in oxidative phosphorylation,PI3K/Akt and MAPK signaling pathway,etc.After the cells in each group were treated with 2μmol/L LY294002,the cell viability was significantly decreased（P<0.05）.Compared with the Control group,the ROS content（P<0.001）,Caspase-3 and Cyt C m RNA（P<0.01）and Bax m RNA（P<0.05）expression in the Control+LY group were significantly increased,and Bcl-2 m RNA expression was significantly decreased（P<0.05）.Compared with the HS group,the ROS content,Cyt C,Caspase-3 m RNA（P<0.01）and Bax m RNA（P<0.05）expression in HS+LY group were significantly decreased,and Bcl-2 m RNA expression was significantly increased（P<0.05）.Compared with the HS+PR group,the ROS content（P<0.01）and Caspase-3 m RNA（P<0.05）in the HS+PR+LY group were significantly decreased.Conclusion:PTD-FNK may alleviate HS-induced boar SCs damage by regulating PI3K/Akt signaling pathway and apoptotic factors,reducing apoptosis,or maintaining mitochondrial function and MMP stability,inhibiting the release of ROS,Cyt C and Ca²⁺,enhancing the antioxidant capacity of cells,or maintaining the integrity of BTB,etc.