Protective Effect Of Resveratrol On Lipopolysacchride-Induced Inflammatory Injury In Porcine Leydig Cells And Its Potential Mechanism

Leydig cells are important specific cells in the testis,which are the main source of testosterone in the testis and involved in spermatogenesis.Immune active factors secreted by the Leydig cells can regulate of the testicular local microenvironment.Lipopolysaccharide(LPS),the main virulence factor of Gram-negative bacteria,induces inflammatory response via activation of the related inflammatory pathways.The inflammatory injury of the Leydig cells affects the reproductive performance of breeding males and the antibiotics therapy is a traditional effective method.With the restriction of antibiotic use,natural plant extracts have attracted more attention.Resveratrol,a natural polyphenolic phytoalexin,has potential biological activities,such as anti-bacteria,anti-tumor,anti-aging and anti-apoptosis etc.In the present study,to investigate the anti-inflammatory effects of resveratrol and its possible mechanism,porcine Leydig cells was induced inflammatory injury by using LPS followed by intervention therapy with RES.The results obtained are as follows:1.When the primary porcine Leydig cells were induced with 5 ?g/mL LPS for12 h,cell viability were significantly decreased by using MTS assay and Real-time iCELLigence system monitoring.The expression of pro-inflammatory factors TNF-alpha and IL-6 was increased,and the expression of anti-inflammatory factors GAS6 and TGF-beta 1 decreased in primary porcine Leydig cells when induced with LPS by RT-qPCR analysis.The data indicated that inflammatory injury could be induced when the primary porcine Leydig cells were treated with 5 ?g/mL LPS for 12 h.2.RES played a certain therapeutic role against the inflammatory injury in the primary porcine Leydig cells caused by LPS.Compared to the porcine Leydig cells,RES treatment to some extent significantly improved the cell viability,reduced the cell apoptosis of the inflammatory-injured cells,and inhibited the expression of TNF-alpha and IL-6,and increased the the expression of GAS6 and TGF-beta 1.Moreover,testosterone level was increased with the RES treatment in inflammatory damaged porcine Leydig cells,with increase of the expression of steroid synthases including StAR,P450 scc,and 3?-HSD.RES treatment promoted the expression of Bcl-2 while inhibited the expression of Bax in the LPS-induced primary porcine Leydig cells.In addition,RES regulated NF-?B and Akt signaling pathways in the inflammatory-injured leydig cells via down-regulation of the phosphorylation levels of p65 and Akt.These data indicate that LPS exerts anti-inflammatory and anti-apoptotic activities at least partially through regulation of NF-?B and Akt pathways.In summary,RES treatment can balance the levels of pro-inflammatory factors and anti-inflammatory factors and enhance the secretion of testosterone in inflammatory-injured porcine Leydig cells.The regulation of RES on LPS-induced inflammatory injury in porcine Leydig cells may be achieved via inhibiting the phosphorylation of some factors in NF-?B and Akt pathways,allowing the Leydig cells to maintain local microenvironment stability.The above results can provide a theoretical basis for the development of RES as a replacement of feeding antibiotics or therapeutic medicine.