Research On Effect And Mechanism Of Kang-fu-ling On Damage Of The Reproductive System Exposed To Microwave Radiation In Male Rats

ObjectiveWith the rapid development of science and technology, microwave technologyhas been widely applied in various fields. Microwave radiation also widely exists indaily life and working environment. Studies show that microwave radiation can causedamage on multiple systems of organism and reproductive organs are one of sensitivetargets. The medical protection of reproductive organs’s injury becomes a hot researchthese years because the reproductive health is related to human happiness and survivalcivilization. However, currently there is a lack of high efficiency, low toxicity, lowprice drug for protecting reproductive system from microwave radiation, sodeveloping the drugs of preventing the damage and promoting the recovery ofreproductive injury has important social and military significance. Therefore, the aimof this study is to investigate the prevention effect, therapeutic effect and effectivedose of traditional Chinese medicine compound preparations namedKang-fu-lingagainst damage in the reproductive system from microwave radiation inrats. On this basis, discuss the reproductive protection mechanism of Kang-fu-lingthrough lipid peroxidation and Catsper1and provide evidence for clinical application.Materials and Methods(1) Prevention Effect of Kang-fu-ling.100male Wistar rats were randomlydivided into five groups: control group(C), radiation group(R), small-dose ofKang-fu-ling group(S), middle-dose of Kang-fu-ling group(M) and larger-dose ofKang-fu-ling group(L),20rats per group. The rats received a daily intragastricadministration from7days before radiation to7days after radiation once a day. Thedosages of Kang-fu-ling in group L, M and S were4g×gn-1×1-1,2g×gn-1×1-1and1g×gn-1×1-1respectively, and the rats in group N and R were given isopyknic distilledwater. The way of radiation was an average power density of30mW/cm2microwaveradiation source with general uniform radiation. Group N on the stage and box did notaccept radiation. The rats in each group were executed at7days of administration (6hours after radiation),6hours after drug withdrawal (7days after radiation),7days after drug withdrawal (14days after radiation) and14days after drug withdrawal (21days after radiation). The rat’s sperm motility parameters were detected by SCAsperm image analysis system. The malformation rate of sperms was calculatedthrough H&E staining. The changes of testicular tissue morphology and ultrastructurewere observed by light and electron microscopy.(2)Therapeutical Effect of Kang-fu-ling.120male Wistar rats were randomlydivided into six groups: control group(C), radiation group(R), anti-radiation medicine(An-duo-lin) group(A), small-dose Kang-fu-ling group(S), middle-dose Kang-fu-linggroup(M) and larger-dose Kang-fu-ling group(L),20rats per group. The rats receiveda daily intragastric administration from the day of radiation to14days after radiationonce a day. The dosages of Kang-fu-ling in group L, M and S were3gng--1×1-1,1.5g5g--1×1-1and0.75g75g-1×1-1respectively, the dosages of An-duo-lin in groupAwere1.5g×.5-1×1-1and the rats in group N and R were given isopyknic distilled water.The way of radiation, the execution time of experimental animal and observationalindex were the same as aforementioned.(3) Protective Mechanism of Kang-fu-ling. Protective mechanism ofKang-fu-lin. At Day7,14to28after radiation, the lipid peroxidation damage indexMDA, antioxidant enzymes activity index SOD and ATP level in the testicular tissueswere detected by microplatereader. The protein and mRNA levels of catsper1weredetected in the group N, R and M using western blot, real-time PCR and imageanalysis technologies.Result(1) Preventive Effect of Kang-fu-lingThe changes of the motion of sperms in rats. At the14day after30mW/cm2microwave radiation, the AR and A plus B sperms of group R were decreasedobviously(p<0.05) compared to the group L, while class D sperms were increased(p<0.05).The changes of the malformation rate in rats’ sperms.The rates of abnormalsperms increased when they were exposed to30mW/cm2microwave radiation after7-14days (p<0.05or p<0.01). The rates of abnormal sperms in group M and Ldecreased compared to group R (p<0.05). Histological changes in the rats’ testis.After30mW/cm2microwave radiation,the seminiferous tubule epithelium was loosed accompanied by degeneration, necrosisand exfoliation of spermatogenic cells. The injury was observed at6hours, the injuryaggravated after7days and it started recovery at the14th day. The injury in group Swas the same as that in group R. While in group M and L the injury was obviouslyrelieved.Ultrastructural changes in the rats’ testis.After7days of radiation withouttherapy in group R, the spermatogonium was edema and intercellular spaces weredilated. Nuclear chromatin of spematogenic cells was agglutinate, side and necrosis.The nuclear membrane was dissolved. The mitochondria were swell and cavitation.The heads of mature sperms were abnormal. While in group M, after7days ofradiation with therapy in group M, the structure of the majority of spermatogonium,spermatocytes and sperms were normal. Slightly swelled mitochondria and abnormalsperms were occasionally found.(2) Therapeutical Effect of Kang-fu-lingThe changes of the motion of sperms in rats. After14days of radiation, the ARand A plus B sperms of group R were decreased obviously (p<0.05) compared to thegroup C, while class D sperms were increased (p<0.05). Sperm motion parametersVCL, VSL and VAP decreased (p<0.05). Compared to group R, the A plus B sperms ofgroup A, M and group L were increased obviously (p<0.05), class C sperms weredecreased (p<0.05), and class D in group M decreased (p<0.05). Sperm motionparameters increased with the increase of AR (p<0.05or p<0.01).The changes of the malformation rate in rats’ sperms. After microwaveradiation7days to14days, the malformation rate of sperms was higher in group Rthan in group C (p<0.05). The malformation rate of group A, M and L decreasedcompared to group R exposed to microwave radiation at administration for7days(p<0.05) and drug withdrawal6hours (p<0.05).Histological changes in the rats’ testis. After30mW/cm2microwave radiation7days, Sperm cells were degenerated, necrosis and fell off. Sperms reduced and proteinand edema fluid accumulated. After14days, the lesions showed a trend of recovery.The injury in group S was the same as that in group R. In group M, there were loosenseminiferous epithelium, a small amount of spermatogenic cells and interstitial edema. The severity in group A, L and M were similar.Ultrastructural changes in the rats’ testis.After7to14days of radiation, thechromatin of spematogenic cells was agglutinate, side and necrosis. The nuclearmembrane dissolved. The spermatogonium was edema and intercellular spaces weredilated. Nuclear chromatin of spematogenic cells was agglutinate, side and necrosis.The nuclear membrane dissolved and intranuclear inclusions were seen. Themitochondria were swell and cavitation. The heads of mature sperms were abnormal.While in group M, the structure of the spermatogenic cells was normal at6hours ofdrug withdrawal. Slightly swelled mitochondria and abnormal sperms wereoccasionally found.(3) Protective mechanism of Kang-fu-lingThe rats’ testis oxidative damage and the change of antioxidant enzymeactivity. After7days to21days of30mW/cm2microwave radiation, MDA in rats’testis tissues was increased (p<0.05). MAD in group M and L decreased compared thatin group R at7days of administration and6hours to7days after drug withdrawal(p<0.05). The SOD activity in testis tissues was decreased after7days to21days of30mW/cm2microwave radiation (p<0.05). The SOD activity in group M and L wasincreased compared to group R (p<0.05).ATP content changes in the rats’ testis.After7days to14days of30mW/cm2microwave radiation, the content of ATP decreased obviously (p<0.05). The content ofATP in group M and L increased after7days of administration compared to group R(p<0.05).Catsper1protein level and mRNA changes in the rats’ testis.After7days to14days of30mW/cm2microwave radiation, the catsper1protein and mRNA levels intestis tissues decreased obviously (p<0.05). While in group M, they were increased at7days of administration and6hours of drug withdrawal (p<0.05).Conclusion(1)30mW/cm2microwave radiation can cause AR of rats’ sperms decrease, rate ofmalformation increase and structural damage in rat’s testis tissues. The degree ofinjury was associated with increase of MDA, decrease of SOD activity and content ofATP and down-regulation of Catsper1protein and mRNA levels. (2) Different dosages of Kang-fu-ling have protective effect on injuries frommicrowave radiation mentioned above. Middle dose and large dose have obviousprotective effect.(3) The effective dose of Kang-fu-ling on treating reproductive injury induced bymicrowave radiation was1.5g/kg/d.(4)Kang-fu-ling can protect injury of rats’ testis tissues from microwave radiationand promote recovery through increasing activity of SOD in rats’ testis tissues,scavenging the over free radical and MDA in testis tissues and inhibiting lipidperoxidation.(5) Kang-fu-ling may suppress calcium overload in spermatogenic cells byregulating protein and mRNA levels of Catsper1and protect the damage ofspermatogenic cells and sperms and promote their recovery.