Inhibitory Mechanism Of EGCG On Acrylamide-induced Testicular Injury In Rats

Acrylamide(ACR)is one of the products of the Maillard reaction and widely found in carbohydrate-rich fried or baked foods.In scientific research and industrial production,ACR is often used as a laboratory reagent and industrial raw material,causing occupational and environmental exposure to humans.In addition,ACR is also found in cigarette smoke.Studies have shown that ACR has clear toxic effects in reproduction,neurology,and genetics.The widespread exposure of ACR to the population poses a serious threat to human health,therefore,exploring how to inhibit the toxic effects of ACR is significant.Studies have shown that ACR has reproductive toxicity and can cause testis tissue damage.Epigallocatechin-3-gallate(EGCG)is one of the main bioactive components of green tea,which has many biological activities such as antioxidant capacity.It has been shown that EGCG can inhibit the neurotoxicity of ACR,but whether EGCG has an inhibitory effect on the testicular toxicity of ACR and its mechanism are unclear.In this study,SD rats were used to establish the ACR injury model to investigate the inhibitory effect of EGCG on ACR-induced testicular injury.The effect of EGCG on ACR-induced testicular toxicity was investigated by recording the body weight and testis index of the rats.The effects of EGCG on ACR-induced pathological changes and cell apoptosis in rat testis tissue were detected by HE staining and TUNEL staining.The effects of EGCG on the expression levels of FSH,LH and PCNA in rat testis were examined by immunohistochemistry.Then,bioinformatics method was used to analyze the data set related to ACR-induced testis tissue damage in the NCBI database,and the differentially expressed genes were screened according to the conditions of adj.P < 0.05 and |log2(Fold Change)| > 1.The protein-protein interaction network was constructed based on the STRING database for the differentially expressed genes,and the MCODE plugin of Cytoscape software were used to derive important functional modules to obtain the key genes.GO and KEGG enrichment analysis of the key genes were conducted to obtain the key genes enrichment signaling pathways of ACR-induced testicular injury and predicted the testicular toxicity mechanism of ACR.On this basis,q PCR and Western Blot techniques were used to validate the key genes and detect the expression of ribosomal protein S9(Rp S9)and its involved in NF-κB signaling pathway-related proteins,ribosomal protein L5(Rp L5)and its involved in ribosomal protein-MDM2-p53 signaling pathway,cell cycle and DNA damage response signaling pathway-related proteins,cytochrome C oxidase 6c(Cox6c)and cytochrome C oxidase 7a2(Cox7a2)and their impact on mitochondrial pathway apoptosis and MAPK signaling pathway-related proteins,to analyze the inhibitory mechanism of EGCG on ACR-induced testicular injury from the perspective of ribosomal injury and mitochondrial injury.The results showed that EGCG could inhibit the significant decrease in body weight and testis index,testis tissue morphology damage,the significant increase in the number of apoptotic cells and the significant decrease in FSH,LH and PCNA expression levels caused by ACR,indicating that EGCG could significantly alleviate the testicular damage induced by ACR.Based on bioinformatics analysis,it was found that the key genes of ACR-induced testis injury mainly involved in biological processes such as ribosome biogenesis,affected cellular components such as mitochondrial protein complexes and ribosomal subunits,and had molecular functions such as composing structural components of ribosomes,and were enriched in signaling pathways such as ribosomes-related signaling pathways,these results suggested that the toxic mechanism of testicular damage caused by ACR may be related to ribosomal damage and mitochondrial damage.The m RNA expression and protein expression levels of Rp S9 and Rp L5 in rat testis after ACR treatment were significantly decreased compared to the Control group.Compared with the ACR group,EGCG significantly upregulated the expression of the above genes and the expression of SENP1,MDM2 and p53 proteins in testis,and significantly inhibited the activation of NF-κB signaling pathway and significantly downregulated the expression of cell cycle regulatory proteins such as Cyclin B1,p-Chk2/Chk2,p-Chk1/Chk1,p21 and DNA damage response-related proteins such asγH2AX/H2 AX,suggesting that EGCG may affect cell proliferation,inflammation,cell cycle regulation and DNA damage response to inhibit ACR testicular toxicity through regulating the expression of Rp S9 and Rp L5.The m RNA expression and protein expression levels of Cox6 c and Cox7a2 in the testis of ACR-injured rats showed a significant decrease compared with the Control group.Compared with the ACR group,EGCG significantly upregulated the expression of the above genes,significantly inhibited the expression levels of mitochondrial apoptosis pathways-related proteins such as Bax/Bcl-2,cleaved caspase-9,cleaved caspase-3and Cyt C,and significantly decreased the phosphorylation levels of proteins related to MAPK signaling pathway,indicating that EGCG may regulate mitochondrial pathway apoptosis and MAPK signaling pathway by affecting the expression of Cox6 c and Cox7a2,in order to exert an inhibitory effect on the testicular toxicity of ACR.The above results suggested that EGCG can effectively improve ACR-induced testis tissue damage in rats,and its mechanism may be related to the inhibition of ribosomal damage and mitochondrial damage.This study demonstrated that EGCG can effectively inhibit the testicular toxicity of ACR,and investigated the inhibition mechanism of EGCG on ACR-induced testicular damage at the animal level,which provides a theoretical basis and relevant experimental basis for reducing the potential hazard of ACR to human health,and also provides support for a more in-depth and comprehensive exploration of the active function of EGCG.