| Background:Male infertility has received global attention in recent years.As an important type II pyrethroid pesticide,cyfluthrin is commonly residing in environmental media and agricultural products due to the increasing use,but there is still a paucity of studies on the correlation between cyfluthrin exposure and testicular function impairment and spermatogenic dysfunction in human and mammals.Therefore,it is urgent to study the effects and mechanisms of action of cyfluthrin exposure on male reproductive health in humans and other mammals.The testis is a sensitive target organ for many drugs and chemicals,and when hormone levels are altered in the testis and other reproductive organs due to environmental endocrine disruptors,it may cause an imbalance in the homeostasis of the reproductive endocrine system and lead to oxidative stress in testicular tissues,which is closely related to male infertility and may be the initiating factor and key link in testicular damage.To find the key genes involved in oxidative stress during testicular spermatogenic dysfunction caused by cyfluthrin will provide new ideas for the subsequent research of male reproductive damage by cyfluthrin.G3BP1 is an important regulator of stress granule formation and function,and stress granule formation can prevent stress-induced cell damage and death.However,the occurrence of testicular spermatogenic dysfunction induced by flucytamethrin by this gene has been less studied,and the specific molecular mechanism is still unclear.Therefore,in this study,we established an in vivo and in vitro model for the development of testicular and germ cell toxicity induced by cyfluthrin,and explored the role and mechanism of G3BP1 gene mediated P38MAPK/JNK pathway in testicular and germ cell damage caused by cyfluthrin to find early and sensitive indicators and new therapeutic targets for the development of testicular damage.Objectives:To establish in vivo model of testicular injury in male rats caused by cyfluthrin and in vitro model of GC-2 spermatocyte injury caused by cyfluthrin.To investigate the role of G3BP1 gene and P38 MAPK/JNK pathway in male reproductive function damage caused by cyfluthrin.To find new biomarker for the early and sensitive indicators of male reproductive injury development and treatment,and also to provide new ideas for the prevention and treatment of male reproductive system diseases.Methods:1.Establishment of in vivo model of testicular injury in male Wistar rats caused by cyfluthrin and in vitro model of GC-2 spermatocyte injury caused by cyfluthrin1.1 Establishment of an in vivo model of testicular injury in male Wistar rats caused by cyfluthrin(1)In vivo injury modelFirstly,40 male Wistar rats(about 260g)were divided into control group(corn oil),low dose group(6.25mg/kg),meddle dose group(12.5mg/kg)and high dose group(25mg/kg).The rats were anesthetized and executed after 28 days of poisoning on alternate days.(2)Observation of pathological and ultrastructural changes of rat testis by cyfluthrinThe body weight of the poisoned rats was monitored daily to observe whether there was any change in the body weight of the poisoned rats,the morphological and ultrastructural changes of germ cells at all levels of testes caused by cyfluthrin were observed by HE staining and transmission electron microscopy.(3)Detection of alteration of male reproductive hormone levelThe effects of cyfluthrin on androgen levels in rats were measured by using Elisa kits for the detection of gonadotropin-releasing hormone,testosterone,serum follicle-stimulating estrogen and luteinizing hormone in serum of rats infected with cyfluthrin.(4)Detection of oxidative stress damage to testes and expression of G3BP1,a key factor of stress granules,by cyfluthrinSOD,T-AOC and MDA kits were used to evaluate the oxidation and antioxidant levels in the post-testis of cyfluthrin-contaminated rats.LDH kits were used to evaluate the expression levels of LDH in the testis of cyfluthrin-exposured rats.q-PCR,Western blot,immunohistochemistry and immunofluorescence were used to detect the effect on the e xpression of G3BP1 gene after cyfluthrin-exposured rats.(5)Detection of testicular inflammation level and ATP changesTo detect the effects of cyfluthrin on androgen levels in rats,Elisa kits were used to test the serum levels of inflammatory factors TNF-α and IL-6 in rats to determine whether cyfluthrin could induce an inflammatory response.ATP kits were used to evaluate the expression levels of ATP in the testis of cyfluthrin-exposured rats.1.2 Establishment of in vitro model of GC-2 spermatocyte damage caused by cyfluthrin(1)Establishment of in vitro modelThe GC-2 spermatocytes were divided into 4 groups: control,low-dose(50μg/m L),middle-dose(100μg/m L)and high-dose(200μg/m L),and the duration of the dyeing was 48 h.(2)Detection of the effect of cyfluthrin on the function of GC-2 spermatocytesThe CCK8 method and scratch assay were used to identify the effects of flucytetramethrin on cell viability and migration ability.The expression level of LDH in spermatocytes after flucytamethrin staining was measured using LDH kit to evaluate its cytotoxic effect.(3)Detection of oxidative stress damage and expression of G3BP1,a key factor of stress granules,in GC-2 spermatocytes after flucytamethrinThe changes of G3BP1 gene expression in spermatocytes after cyfluthrin staining were detected by q-PCR,Western blot,immunohistochemistry and immunofluorescence.The changes of ROS content in spermatocytes after cyfluthrin staining were evaluated by flow cytometry,and the oxidation and antioxidant levels in spermatocytes after cyfluthrin staining were evaluated by SOD,T-AOC and MDA kits.(4)Detection of the alteration of GC-2 spermatocytes inflammation level and ATP levelThe levels of inflammatory factors TNF-α,IL-6 in the serum of spermatocytes of dyed rats were examined by q-PCR and Western blot,and the expression levels of ATP were evaluated to assess the mitochondrial damage.1.3 Detection of changes in the expression of key factors of MAPK pathway in vivo model of testicular injury and in vitro model of GC-2 spermatocyte injury caused by cyfluthrinBased on the establishment of the previous in vivo and in vitro male reproductive injury models,q-PCR and Western blot were further used to examine the expression of MAPK pathway key factors(p-JNK1/2/3,JNK1/2/3,p-ERK1/2,ERK1/2,p-P38 MAPK,P38MAPK),mitochondrial respiratory chain enzyme complexes(COX1,COX4),apoptotic factors(Caspase3,Caspase8,Caspase9)were examined for changes in m RNA and relative protein expression levels,and the TUNEL method was used to detect chlorpyrifos-induced spermatocyte apoptosis and further elucidate the role of G3BP1 gene and P38 MAPK/JNK pathway in the male reproductive toxicity caused by chlorpyrifos staining.1.4 Regulation of G3BP1 gene expression and observation of its role in GC-2spermatocyte injury model caused by cyfluthrin(1)Establishment of a lentiviral vector to inhibit G3BP1 expression and G3BP1overexpressionIn order to further clarify the role of G3BP1 gene and P38 MAPK/JNK pathway in the male reproductive toxicity caused by cyfluthrin,a model of male reproductive damage in GC-2 spermatocytes by disrupting and overexpressing G3BP1 gene was constructed by lentiviral transfection.The expression of G3BP1 gene at m RNA and protein levels was detected by puromycin drug sieve,fluorescence microscopy,as well as WB and q-PCR to determine whether the G3BP1 gene overexpression and inhibition of expression cell models were successfully established.(2)Detection of the effects on GC-2 spermatocyte function after overexpression and inhibition of G3BP1 expressionThe changes of cell damage and viability after overexpression and inhibition of G3BP1 gene expression were detected by CCK8 method;the changes of LDH expression level in spermatocytes after overexpression and inhibition of G3BP1 gene expression were detected by LDH kit.(3)Detection of the effect of fluthrin on the level of oxidative stress in GC-2spermatocytes after overexpression and inhibition of G3BP1 expressionSOD,T-AOC and MDA kits were used to detect the changes of oxidative and antioxidant levels in cells after overexpression and inhibition of G3BP1 gene expression;red fluorescence detection kits were used to detect the corresponding changes of ROS in spermatocytes after overexpression and inhibition of G3BP1 gene expression.(4)Detection of the effects of the inflammation level and ATP in GC-2spermatocytes after G3BP1 overexpression and inhibition of expressionThe changes of ATP content in stained spermatocytes after overexpression and inhibition of G3BP1 gene expression were detected by ATP assay kit,and the inflammatory factors TNF-αand IL-6 content were detected by q-PCR and Western blot to assess their effects on inflammation levels and mitochondria.(5)Detection of the effect of the expression of key factors of MAPK pathway in GC-2 spermatocytes after G3BP1 overexpression and inhibition of expressionFurther,q-PCR and Western blot were used to detect the changes of m RNA and protein relative expression levels of MAPK pathway key factors and their phosphorylated proteins,mitochondrial respiratory chain enzyme complexes and apoptotic factors in stained spermatocytes after overexpression and inhibition of G3BP1 gene expression,to investigate the effects of regulating G3BP1 gene expression on MAPK pathway and its possible induced alterations in the cascade response.Results:1.Establishment of in vivo model of testicular damage in male Wistar rats caused by cyfluthrin and in vitro model of GC-2 spermatocyte damage caused by cyfluthrin1.1 Establishment of in vivo model of testicular injury in male Wistar rats caused by cyfluthrin(1)Cyfluthrin could cause pathological and ultrastructural changes in the testisThe model of testicular injury caused by cyfluthrin was established.Firstly,the pathological changes were observed by HE staining,and the results showed that the number of cell layers gradually decreased with the increase of the dyeing dose,the morphology of seminiferous tubules changed,the cells in the lumen gradually loosened,the lumen was vacuolated,the sperm density became less and the sperm morphology was incomplete.The results of transmission electron microscopy showed that the edema of the spermatocytes increased with the increase of the dose of poisoning,the cell membrane was locally broken,the mitochondrial cristae were broken and disappeared,and the number of lipid droplets increased,indicating that the cell damage gradually increased with the increase of the dose of poisoning.(2)Cyfluthrin could cause hypergonadotropic dysfunction in ratsThe results of androgenic hormone assay showed that serum Gn RH,FSH and LH levels increased with increasing dose of poisoning,and T levels decreased in a dose-dependent manner,and T/LH decreased significantly,indicating that cyfluthrin could interfere with the normal secretion of hypothalamic-pituitary-gonadal axis and cause hypergonadotropic gonadal dysfunction.(3)Cyfluthrin inhibited the expression of G3BP1 and caused oxidative stress and inflammation in testicular tissueSerum inflammatory factors IL-6 and TNF-α increased with increasing dose,LDH and MDA increased in a dose-dependent manner,and SOD,T-AOC and ATP decreased in a dose-dependent manner,indicating that inflammation occurred in the organism and the oxidative-antioxidative homeostasis was disrupted.The results of Western blot,q-PCR,immunofluorescence and histochemistry showed that the relative m RNA and protein expression of G3BP1 decreased with increasing dose of toxicity,and the positive expression of G3BP1 was mainly in the testicular germinal epithelium and germ cell layer.The positive expression of G3BP1 was mainly located in the epithelium and germ cell layer of testis.1.2 Establishment of an in vitro model of GC-2 spermatocyte damage caused by cyfluthrin(1)Cyfluthrin caused changes in morphology,viability and migration ability of GC-2 spermatocytesIn the model of spermatocyte damage caused by cyfluthrin,the effect of cyfluthrin on cell viability was firstly examined by CCK8,and the results showed that the cell viability decreased significantly with the increase of the dosing concentration.It was further observed that the cell morphology changed with increasing dose of cyfluthrin,and the number of viable cells in the high dose group decreased significantly,and the cell morphology changed severely.At the same time,the cell migration rate decreased and LDH content increased with the increase of dose,which further caused cell membrane damage and affected cell viability and function.(2)Cyfluthrin can disrupt the oxidation-antioxidation system of GC-2spermatocytes and inhibit the expression of G3BP1With the increase of cyfluthrin dose,the MDA and ROS contents in GC-2spermatocytes gradually increased,while SOD,T-AOC and ATP decreased in a dose-dependent manner.The above results indicated that cyfluthrin could lead to the disruption of oxidative-antioxidant system in spermatocytes leading to oxidative stress.Western blot,immunofluorescence and q-PCR results showed that the expression of G3BP1 decreased in a dose-dependent manner with increasing doses of cyfluthrin.This indicates that the expression of G3BP1,a key component of stress granules,was inhibited with increasing doses of cyfluthrin,and the antioxidant capacity of testicular tissues and spermatocytes was decreased and oxidative stress damage was exacerbated.1.3 Investigation of the role of G3BP1 gene-mediated P38 MAPK/JNK pathway in testicular and spermatocyte dysfunction caused by cyfluthrinThe previous results showed that cyfluthrin induced an imbalance of oxidative-oxidative homeostasis in testis tissue and spermatocytes,and the expression of G3BP1,a key component of stress granules,was inhibited.ERK protein and m RNA expression decreased,p-JNK1/2/3,p-P38 MAPK protein expression increased,COX1 and COX4 showed a dose-dependent decrease,Caspase 3,Caspase-8 and Caspase-9 increased,and the number of TUNEL-positive cells increased with the increase of the dye dose,indicating that when G3BP1 expression is inhibited,it can cause activation of JNK1/2/3,P38 MAPK signaling,interfere with the normal secretion of extracellular signal-regulated kinase ERK,cause mitochondrial dysfunction and activation of dual apoptotic pathways in germ cells,and cause apoptosis of germ cells.1.4 Exploring the role of G3BP1 gene expression in spermatocyte damage after intervention with cyfluthrin(1)Overexpression of G3BP1 alleviated cytotoxicity induced by cyfluthrin,including the degree of cell membrane damage,oxidative stress,and mitochondrial damageG3BP1 gene overexpression lentiviral vector and G3BP1 gene inhibition lentiviral vector were constructed,and it was found that compared with the control and inhibition groups,the LDH and MDA contents of spermatocytes were significantly reduced and the SOD and T-AOC contents were significantly increased after G3BP1 overexpression,and the opposite result was found after G3BP1 inhibition.It showed that overexpression of G3BP1 could alleviate the cytotoxicity induced by cyfluthrin,and the degree of cell membrane damage and oxidative stress were alleviated.Moreover,the relative fluorescence intensity of ROS and ATP content in the stained cells were relatively reduced after overexpression of G3BP1,indicating that overexpression of G3BP1 could alleviate the production of ROS in cytotoxicity induced by cyfluthrin in spermatocytes,inhibit the decrease of ATP to a certain extent and alleviate the mitochondrial damage.(2)Overexpression of G3BP1 inhibited the activation of P38 MAPK/JNK pathway and attenuated the intracellular apoptotic cascade responseWB and q PCR results showed that JNK1/2/3 protein and m RNA expression increased,p-P38 MAPK protein expression decreased,ERK protein and m RNA expression decreased,while p-ERK protein expression increased significantly.The opposite was true after inhibition of expression.The expression of IL-6 and TNF-αprotein and m RNA were significantly decreased,the expression of COX1 and COX4 protein and m RNA were significantly increased,and the expression of Caspase 3,8and 9 protein and m RNA were significantly decreased in the stained cells after overexpression of G3BP1,and the opposite was true after inhibition of expression.This further confirmed that overexpression of G3BP1 could inhibit the activation of P38 MAPK/JNK pathway,alleviate cytosolic damage induced by cyfluthrin and reduce the intracellular apoptotic cascade response.Conclusion:1.Exposure to cyfluthrin could cause testicular and spermatocyte damage in rat testes,which could cause pathomorphology,alterated in androgen levels,decreased antioxidant capacity and inflammation.2.When intracellular antioxidant capacity is impaired,G3BP1 expression and activity were inhibited,causing activation of the P38 MAPK/JNK pathway and activation of the dual intracellular apoptotic pathway,which in turn leaded to germ cell apoptosis.3.After interfering with G3BP1 gene expression,over-expression of G3BP1 significantly alleviated the oxidative stress,inflammation and mitochondrial damage,and apoptosis of spermatocytes induced by flucytethrin through regulating the P38MAPK/JNK pathway.The opposite result was observed after inhibition of its expression.Therefore,the G3BP1 gene could be involved in the oxidative stress,inflammation,mitochondrial damage and apoptosis of testis and spermatocytes caused by cyfluthrin through the regulation of P38 MAPK/JNK pathway,and thus affected the occurrence and development of male reproductive function damage by cyfluthrin. |
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