| Exercise-induced Fatigue is the overload continuous movement to makethe body function and exercise load incompatible, resulting in fatiguecontinuous accumulation, giving rise to a series of functional disorders orpathological state, or fatigue associated with health damage caused by themovement of the body’s physiological and biochemical changes resultinginathletic ability of the body temporarily reduced. Traditional Chinesemedicine has a unique advantage in delay and eliminate fatigue,TraditionalChinese medicine has a unique advantage in delay and eliminatefatigue.Heshouwuyin uesd in this study was made by Polygonum multiflorumpill in “Vision on the side”,including heshouwu,Cistanche,Achyranthes,danshen and blood,longevity,elininating the effect of fatigue.Inthis study,heshouwuyin was ued to treat of rats with sports fatigue which wereobserved blood biochenical indices and testosterone synthesis rat-limitingenzyme,P450scc and STAR,as testicular cell cholesterol synthesis rats-limitingenzyme HMG-CoA and SR-BI expression.Lay the theoretical andexperimental basis for further study the effect and mechanism of the Chinesemedicine anti-sports fatigue.Objective:Biochemistry,immunohistochemistry,RT-PCR and Western Blottingmethod were used to detect the blood biochemical indices as well as theP450scc,of STAR and HMG-CoA,the SR-BI expression in testicular tissue ofrats in each group.Explore the mechanism of Heshouwuyin anti-sports fatigue.Method1. The choice of experimental animals and groups: We used Seventy-fivemale SD(Sprague-Dawley, SD) rats who were all Eight-week-old.（Animalqualified certificate serial number1005050.） They body weight180~200g。freely take a meal, drinking, Indoor temperature20℃-25℃, The relative humidity is45%-55%, only shine on every day12h，75SD rats wererandomly divided into6groups, including normal control group(A group),heshouwuyin administered normal group(B group)，model control group(Cgroup), sponsored recovering group（D group），heshouwuyin treated group (Egroup), heshouwuyin prevented group (F group), each group15animals.2. Establishment of a rat model of exercise-induced fatigue: Theswimming pool wall smooth, and the inner wall of a circular, and the diameterwas50cm, and the depth was70cm. The water temperature was30℃±2℃.We made the rats to swim adaptively at the first week.Once a day,a total of7times.The first day,swim in the water for30minutes, and then the timeincreased by40%daily until the rats could swim for2hours at the firstweekend. The adaptive weight loading swimming training began at the secondweek. The rats burdened with0.5%of their body weight for the first time,andthe weight increased by1%、2%、3%、4%daily. The rats swam for2hourseach time,once a day. The load increased to5%of their body weight at thesixth day, and the swimming time was significantly shorter, so we chose4%of their body weigh as the ultimate load.Since then six weeks once a day forthe exhaustion swimming experiments(Exhaustive swimming standards forthe rat to the apparent lack of coordination, can not go up10seconds after rhewater sinking.placed on the plane unable to complete the righting reflex.)3. Each group treatment: Group A rats not to swimming training,andlavaged the same volume of Sodium Chloride; Group B rats not to swimmingtraining, and lavaged the same volume of Sodium Chloride andHeshouwuyin(20g/kg.d). Group C, group D,group E and group F didswimming training every afternoon to copy exercise-induced fatigue model.Group F was fed with Heshouwuyin (20g/kg/d) every morning for theprevention and swam in the afternoon for6weeks. We collectd about1.5mlthe medial canthal orbital vein blood. After42days since we setted up themodels, The blood was centrifugated,and we drew on the supernatant. Wedetectd the concentration of blood serum testosterone,blood urea nitrogen,blood lactate, blood hemoglobin and the blood serum creatinine. Then we judged the modeling is successful or not. The judging standard is as following:Compared with quiet control group, the concentration of blood urea nitrogenand blood lactate increased significantly in model group rats,rats haddecreased appetite weight loss,which indicatd that the modeling wassuccessful. After the success of the model,C group of rats were sacrifilced;Group D rats freely take a meal, drinking,not given any intervention; Group Erats was given Heshouwuyin (20g/kg/d) for treatment everyday; Group F ratsdiscontinuation of treatment stop training, freely take a meal, drinking,60daysin a row4.specimen collection and treatment:After the end of the experiment, Ratsin each group were anesthetize with10%chloral Hydrate. We collectd about1.5ml the medial canthal orbital vein blood, the blood was centrifugated for20minutes at a speed of1000r/min, and then we drew on the supernatant, andthe supernatant was keep at-80℃refrigerator. Next from left ventricle to theaorta, and then we perfused approximately250ml Sodium Chloride fast whichwas treated with DEPC until the flowing liquid was clear. Then we took theleft testicular tissue quickly.We kept the tissue in liquid nitrogen for30minutes, and took the tissue to-80℃refrigerator for keep to spare for theWestern blotting, RT-PCR detection. The right testicular tissue was fixed with4%paraformaldehyde by perfusion, and the tissue was embedded in paraffinin order to spare for immunohistochemistry.Results:1.General state of health to observe:The rats of group a and b fur cleanand tidy,freedom of movement and good spirits.The remaining four groups ofrats the universal spirit of fatigue,bradykinesia,indifferentmfur loss,emaciatedbody,diet loss,weight loss.2.The concentration of serum testosterone and hemoglobin: Comparedwith quiet control group，the concentration of testosterone in model controlgroup decreased, hemoglobin content decreased there was significantdifference (P<0.05). Compared with model control group the concentrationof serum testosterone in Heshouwuyin treated group and prevented group increased significantly(P<0.05)Compared with treatment group，there was nosignificant difference in the concentration of serum testosterone andhemoglobin in prevention group (P>0.05).3.The concentration of blood lactic acid, serum creatinine and blood ureanitrogen: Compared with quiet control group，the concentration of blood lacticacid, serum creatinine and blood urea nitrogen in model control groupincreased significantly (P<0.01). Compared with model control group theconcentration of blood urea nitrogen in Heshouwuyin treated group andprevented group become significantly lower(P<0.05). Compared withtreatment group，there was no significant difference in the concentration ofblood lactic acid, serum creatinine and blood urea nitrogen in preventiongroup (P>0.05).4.immunohistochemistry result: p450scc Immunohistochemical positiveparticles of brown particles was mainly expressed in the Mesenchymal cellsCytoplasm and Spermatocyte cytoplasm, group B, group E and group Fpositive signal was stronger than group D and group A (P <0.05), group A wasstronger than group C (P <0.05). HMG-CoA immunohistochemical positiveparticles were mainly expressed in the testicular interstitial cells of group Band group F, expression of the weakest group C of the strongest, statisticallysignificant compared with the other groups. STAR and SR-BIimmunohistochemistry positive particles are mainly expressed in the Leydigcell cytoplasm, group B, group E and group F, positive signals were strong ingroup A and group D (P <0.05), Group C positive signal is the weakest.5.Western blotting and RT-PCR results:P450scc protein and mRNAexpression changes as follows:Group B, group E and group F wassignificantly higher than that of group D and group A (P <0.05), group C wassignificantly lower in group A (P <0.05), no significant difference betweengroup A and group D; of HMG-CoA protein and mRNA expression changes asfollows: group B and group F was significantly lower than in Group E, groupD and group A (P <0.05), group C was significantly higher in group A (P <0.05)between group A and group D, no significant difference; STAR and the SR-BI protein and mRNA expression changes as follows: group E and group F wassignificantly higher than in group A and group D (P <0.05), group C wassignificantly lower in group A (P <0.05) in group A and D no significantdifference between groups.Conclusion1.The rat model of exercise-induced fatigue is successful.2.Heshouwuyin can increase the concentration of serum testosterone andhemoglobin.3.Heshouwuyin can significantly reduce the concentration of blood lacticacid, serum creatinine and blood urea nitrogen of exercise-induced fatiguerats.4.Heshouwuyin by reducing HMG-CoA and to improve the SR-BIprotein expression, and improve exercise fatigue testosterone secretion in rats.5.Heshouwuyin can improve testosterone biosynthesis rate-limitingenzyme P450scc and StAR expression. |
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