Effect Of Cuscuta Flavone On The Apoptosis Of Mouse Leydig Cells Induced By Bisphenol A

Environmental endocrine disruptor of Bisphenol A(BPA),which is widely used as a plasticizer,including baby plastic bottle and metal material food and beverage container inner wall coating braces sealant.Because BPA has a chemical structure similar to that of estrogen,it has weak estrogen and strong antiandrogenic activity,leading to male reproductive developmental toxicity.As an active ingredient in the kidney-removing Chinese medicine,the flavonoids of Cuscuta chinensis not only have the function of immunomodulating and anti-oxidation,but also improve the function of reproductive organs.Therefore,this study used testicular stromal cells as the research object,and observed the changes of related indicators to illustrate the effect of dodder flavonoids on the reproductive toxicity of BPA.In vivo test: 60 Kunming male mice were randomly divided into 5 groups,negative control group,BPA group and flavonoids of the Cuscuta chinensis(10 mg/kg),medium(20 mg/kg),and high(30 mg/kg)Dose protection group.The model group was given BPA at 20 mg/kg every day.The protective group was also given the corresponding dose of flavonoids and BPA(20 mg/kg)once daily for 7 days.At the end of the 7th day,blood was collected from the eyeball and sacrificed.Blood samples were separated and the levels of testosterone and luteinizing hormone in serum were measured by radioimmunoassay.Testicular sections were prepared,sperm motility was detected by light microscopy,staining localization and purity analysis of Leydig cells were performed.The number of apoptotic bodies was detected by TUNEL method.The transcription levels of ERK,P38 and JNK in testicular tissue interstitial cells were detected by Real-Time Q PCR.The expression of MAPK pathway protein phosphorylation was detected by Western Blotting.The results showed that compared with the negative control group,BPA at 20 mg/kg significantly reduced the levels of testosterone and luteinizing hormone in the serum of mice(P<0.01);compared with the model group,10,20,30 mg/kg.The flavonoids of Cuscuta chinensis could significantly increase the content of luteinizing hormone in serum(P<0.01).The flavonoids of the Cuscuta chinensis of 30 mg/kg can significantly increase the content of testosterone in serum(P<0.01).Compared with the negative control group,the mRNA expression of ERK in the model group was significantly decreased(P<0.05),and the mRNA expression levels of P38 and JNK were significantly up-regulated(P<0.05);compared with the model group,10,20,30 mg/kg flavonoids group significantly up-regulated the expression of ERK mRNA(P<0.05),and the mRNA expression levels of P38 and JNK were significantly down-regulated(P<0.05).Compared with the blank control group,the number of Leydig cells and the number of spermatozoa in the model group were significantly decreased(P<0.05).Compared with the model group,the Leydig cells of the 10,20,30 mg/kg testis stromal cells the number and the number of spermatozoa increased significantly(P<0.05).The rate of sperm deformity decreased significantly with the increase of the concentration of flavonoids(P<0.05).The number of apoptosis of Leydig cells in the model group was significantly higher than that in the control group by TUNEL method(P<0.05).Compared with the negative control group,the phosphorylation of ERK protein in the Leydig cells of the BPA model group was significantly decreased(P<0.01);the phosphorylation of P38 and JNK protein was significantly increased(P<0.01);compared with the model group,each dose group The flavonoids of Cuscuta chinensis significantly increased the expression of ERK protein phosphorylation in Leydig cells(P<0.01),and inhibited the expression of JNK and P-38 protein phosphorylation(P<0.01).High,medium and low doses of flavonoids can significantly alleviate the abnormalities of the above indicators(P<0.05 or P<0.01),and the mitigation effect is positively correlated with the concentration.In vitro test: The mouse testicular stromal cells in good growth state and in the logarithmic growth phase were screened by MTT method to determine the optimal dose of BPA on mouse Leydig cells and the flavonoids of the Cuscuta chinensis on the cells.Safe dose range.In this experiment,the cells were pretreated with a certain dose of dodder flavonoids for 4 h.After BPA interfered with the cells for 24 h,the levels of testosterone and luteinizing hormone in the supernatant were detected by radioimmunoassay.The number of apoptotic bodies was detected by TUNEL method.Real-time PCR was used to detect the transcription levels of ERK,P38 and JNK in BPA-induced mouse Leydig cells.Western Blotting detects the expression of MAPK(mitogen protein kinase)pathway protein phosphorylation.The results showed that compared with the blank control group,BPA at 80 μmoL/mL significantly decreased the secretion of testosterone and luteinizing hormone from Leydig cells(P<0.05).Compared with the model group,the flavonoids group of 250,300,350,400 μg /mL significantly increased the levels of testosterone and luteinizing hormone(P<0.05).The number of apoptotic bodies in the model group was significantly higher than that in the blank control group by TUNEL method(P<0.05).Compared with the BPA group,different concentrations of flavonoids could significantly reduce the number of apoptotic bodies(P<0.05).Compared with the blank control group,the expression of ERK mRNA in the model group was significantly decreased(P<0.05),and the mRNA expression levels of P38 and JNK were significantly up-regulated(P<0.05);compared with the model group,250,300,350 μg/mL The expression of ERK mRNA was significantly up-regulated in the 400μg/mL flavonoids group(P<0.05),and the mRNA expression levels of P38 and JNK were significantly down-regulated(P<0.05).Compared with the blank control group,the phosphorylation of ERK protein in the Leydig cells of the BPA model group was significantly decreased(P<0.01);the phosphorylation of P38 and JNK protein was significantly increased(P<0.01);compared with the model group,the flavonoids of Cuscuta chinensis Significantly increased the expression of ERK protein phosphorylation in Leydig cells(P<0.01),and inhibited the expression of JNK and P-38 protein phosphorylation(P<0.01).250,300,350,400 μg/mL of flavonoids can significantly alleviate the abnormalities of the above indicators(P<0.05 or P<0.01),and the mitigation effect is positively correlated with the concentration.All of the above,the flavonoids of Cuscuta chinensis can alleviate BPA to accelerate apoptosis of testicular stromal cells through MAPK pathway,reduce BPA damage to Leydig cells,and alleviate hormone secretion abnormalities.