| Background:With the using of Cyclophosphamide(CP) widely, the toxic effect ofcyclophosphamide, including the effect on children's reproductive functionand how to protection, is gradually caused the pepole's attention,.Cyclophosphamide,a precursor of the alkylating agents, has no biologicalactivity,except that it was metabolized in the body. Acrolein(ACR), themetabolite of CP, is the main toxic factor of CP. Over the years, someresearchs about the toxicity of alkylating agent on testiS are mainlyrestricted to the observation of tissue morphology. In the preliminary studyof our research group,We found that cyclophosphamide can induce theseminiferous epithelium thinning and the distribution of Sertoli cellsrarefaction,which suggested that the blood-testis barrier (BTB) has beendamaged, but its mechanism is unclear.Now how does thecyclophosphamide damage the blood-testis barrier is still the hot anddifficult question for study.Objective: To investigate the effects of CP on the tight junction and cytoskeletonof Sertoli cell in immature SD rat testis. Intraperitoneal injection of CP invivo and sertoli cells primary cultured in vitro. And on this basis,weexplore the protection role of antioxidants-ginkgetin on sertoli cellsdamaged by acrolein-induced oxidative in vitro,which will provid atheoretical foundation for further clinical application.Methods:①The model of CP induced rats'immature testicular injury wasadopted in vivo. Electron microscopy was used to observe themorphological changes of testicular tissue.The expression of Occludin andZO-1proteins were detected by immunohistochemical and Western Blottechnology.And the expression of F-actin,a cytoskeleton protein,wasdetected by immunofluorescence, and Preliminary study mechanism of CPdamage the blood-testis barrier in vivo.②Sertoli cells was primary cultured in vitro. Cell viability determinedby the MTT method; the change of the main antioxidant enzyme in Sertolicells were detected by Spectrophotometric determination; Measurement ofsuperoxide anion levels by DHE; Ultrastructure changes were detected bytransmission electron microscope; F-actin was detected by Cytochemicalstaining; and the apoptotic index of sertoli cells was detected by flowcytometry,and Western Blot analysis was performed for p38MAPKexpression. To explore the possible mechanism of injury induced by ACR in vitro.③Sertoli cells were pretreatment with ginkgetin before exposure toACR, the expression of F-actin was detected by immunofluorescence, theapoptotic index of sertoli cells was detected by flow cytometry andWestern Blot analysis was performed for p38MAPK expression. To explorethe protective effect of ginkgetin on sertoli cells injured by ACR in vitro.Results:1. Tight-junction in rat's testis injured by CP in vivo:①The control group showed that the junctions of Sertoli cell wassmooth and clear,and walking nature; while treatment group showed thejunction separation, which appear bigger gap, and the endoplasmicreticulum closed to junction also appears in different degree of expansion.②I mmunohistochemical detection showed that there were noSignificant difference for the expression of Occludin and ZO-1betweencontrol group and treatment group(P>0.05).③Immunofluorescence detection showed that there was a Significantdifference for the expression of F-actin between control group andtreatment group(P<0.05)。④The expression of F-actin analysised by Western Blot in controlgroup was decreased more obviously than treatment group(P<0.05),Butthere were no Significant difference for the expression of Occludin andZO-1between control group and treatment group(P>0.05). 2. Sertoli cells injury of ACR in vitro:①Sertoli cells were isolated successfully. Its appearance wasadherence, long fusiform, and2-3protrusions. The purity of Sertoli cellcultures was found to be more than90%.②Effects of ACR on the viability of Sertoli cells were evaluated byMTT analysis. The results show that the survival rate (%) of Sertoli cellswas about68.39±2.1and52.10±1.0at3and12h, respectively,Whenexposed to100lM ACR. These changes were significantly different(P<0.01).③Treatment of Sertoli cells with ACR (100uM) induced a significantdecrease in T-AOC, and SOD, CAT, and GSH-PX activities as comparedwith the control group(P<0.05).④ACR treatment enhanced thered fluorescence of Sertoli cells withthe increased incubation time, indicating that ACR increases ROS levels.⑤Sertoli cells incubated with ACR(100uM) for12h exhibitedprominent aggregation, marginalization and regionalization ofmicrofilaments, a small morphology and rearrangement of the actincytoskeleton.⑥Ultrastructure changes were detected by transmission electronmicroscope in Sertoli cells treated with100uM of ACR for12h, includingmitochondrial swelling, aggregated chromatin, condensed cytoplasm,nuclei splitting and nuclei vacuolization. ⑦FCManalysis indicated that the apoptosis was significantlyincreased compared to those of cells in the control group(P<0.05). Inaddition, p38MAPK expression levels were significantly increased in cellstreated with ACR for12h compared with cells treated for3h(P<0.05)。⑧The expression of p38MAPK analysised by Western blot wassignificantly increased in cells after treated with ACR for3and12hcompared to those of cells in the control group. These data suggest that theexpression of p38MAPK increases in response to ACR treatment in a time-dependent manner.3. Protect role of Ginkgetin on Sertoli cells exposured to ACR:①Adding Ginkgetin before exposed to ACR, FCManalysis indicatedthat the apoptosis was significantly decreased compared to those of cellstreated with ACR alone.(P<0.05)。②Adding Ginkgetin before exposed to ACR, the damage of Sertolicell cytoskeleton was reduced apparently, which closed to the normaldistribution.③Adding Ginkgetin before exposed to ACR,the expression ofP38MAPK was decreased significantly compared to those of cells treatedwith ACR alone.(P<0.05).Conclusion:①Damage Effects of cyclophosphamide on the tight-junction inblood-testis barrier was definite. ②Cyclophosphamide may interfere with the cytoskeleton and induceapoptosis to disrupt the stability of blood-testis barrier.③Cyclophosphamide has no direct effect on the expression oftight-junction protein.④Adding Ginkgetin before exposed to ACR can reduce the damage ofcytoskeleton and apoptosis. Ginkgetin protected sertoli cells probably bydecreasing intracellular ROS levels and inhibition of P38MAPK activation. |
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