The Screening And Mechanism Of Cistanche's Anti-fatigue And Promoting Reproductive Effects To Nourish The Kidney Yang And Its Mechanism

Aim:Kidney-yang deficiency is a type of symptoms of cold caused by the decline of kidney yang and energy.The clinical manifestations are sore waist and knees,loss of libido,impotence,etc.,resulting in the decline of male reproductive function,which is related to the chronic fatigue syndrome and male sexual dysfunction in modern medicine.Cistanches Herba has become one of the first-selected medicines for tonifying kidney due to its functions of invigorating kidney yang and benefiting essence and blood.It is also the most frequently used traditional Chinese medicine in ancient Chinese prescriptions for increasing strength.Modern pharmacological studies have shown that Cistanche tubulosa(Schenk)R.Wight(C.tubulosa)can promote the release of related hormones in the body by regulating the function of the hypothalamus-pituitary-target gland axis,maintain the body's secretory homeostasis,improve immunity and fatigue,and then improve the reproductive capacity of patients.Therefore,this experiment starts with the chronic fatigue syndrome and male sexual dysfunction,which are closely related to kidney-yang deficiency.Through the establishment of two kinds of fatigue models of central and sports and reproductive injury models of rats and mice,the method of combining behavioral observation and the detection of related hormone levels in the circulation was used to screen the pharmacodynamic activity of oligosaccharides of C.tubulosa(Oligose),polysaccharides of C.tubulosa(Glycan)and phenylethanol glycosides from C.tubulosa(CPhGs).And further combined with in vivo and in vitro experiments to initially explore the potential active mechanism and therapeutic characteristics of its main active parts to promote reproduction,to provide an experimental basis for the disclosure of C.tubulosa and subsequent drug development.Methods:1.Screening of anti-fatigue effect parts of C.tubulosaThe central fatigue experiment:The mice were randomly divided into Control,sleep deprivation(SD),Oligose,Glycan,CPhGs groups.A central fatigue model was prepared using sleep deprivation for 48 hours,followed by an eight-arm labyrinth and dark avoidance experiment to test the learning and memory abilities of the mice,and radioimmunoassay was used to determine serotonin 5-hydroxytryptamine(5-HT),norepinephrine(NE),epinephrine(E)in the cerebral cortex and hypothalamus,as well as plasma adrenocorticotropic hormone(ACTH),corticotropin releasing hormone(CRH),cortisol(CORT)levels.The exercise fatigue experiment:The mice were randomly divided into Control,forced swimming(FS),Oligose,Glycan,CPhGs groups.The exercise fatigue model was prepared by forced swimming for 9 days,and then the mice were subjected to a weight-bearing swimming exhaustion experiment to evaluate the endurance,and the liver glycogen,malondialdehyde(MDA),superoxide dismutase(SOD)in the liver were measured,simultaneous determination of muscle glycogen,succinate dehydrogenase(SDH),lactic acid(LD),blood urea nitrogen(BUN),lactate dehydrogenase(LDH),MDA levels.Based on the above methods,the anti-fatigue effects of three different extraction parts of C.tubulosa are screened.2.Screening of promoting reproduction effect parts of C.tubulosaIn order to facilitate the collection of the relevant characteristics of kidney-yang deficiency,rats were used as experimental objects.The rats were randomly divided into Control,reproductive injury model(Model),Oligose,Glycan,CPhGs groups.Intramuscular injection of hydrocortisone for 14 days to prepare a rat model of reproductive injury,measuring the erection latency,mating ability indicators(capture/ejaculation latency,the number of captures/ejaculations),organ wet weight and seminal plasma fructose content.Measure the plasma cyclic adenosine monophosphate/cyclic guanosinc monophosphate(cAMP/cGMP)value,serum gonadotropin-releasing hormone(GnRH),follicle-stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E2)and testosterone(T)levels.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of testis.Based on the above methods,the effects of three different extracting parts of C.tubulosa on reproductive damage were screened.3.Efficacy evaluation of different doses of CPhGs for promoting reproductionIn order to save the amount of drugs and consider the comprehensiveness of the types of antibodies,mice were selected as experimental subjects.The mice were randomly divided into Control,Model,positive drug testosterone propionate(TP),and low-dose of CPhGs(CPhGs-1),middle-dose of CPhGs(CPhGs-m),high-dose of CPhGs(CPhGs-h)groups.Hydrocortisone was intragastrically administered for 14 days to prepare a mouse model of reproductive injury,and the erection latency,mating ability indicators(capture/ejaculation latency,the number of captures/ejaculations),wet weight of organs,serum hormone LH and T levels were detected.And pathological changes of testis were observed by HE staining.Based on the above methods,the efficacy of CPhGs in low,middle and high levels was evaluated from the aspects of testis morphology and mouse reproductive function.4.In vivo and in vitro mechanisms of CPhGs to increase T level and promote reproductionWestern blot(WB)method was used to detect the steroidogenic acute regulatory protein(StAR),cytochrome P450 cholesterol side chain cleavage enzyme(P450scc/CYP11 A1)and 3?-hydroxysteroid dehydrogenase(3?-HSD)expression levels in the T synthesis pathway of testis tissue.The immunofluorescence staining(IF)was used for qualitative and quantitative analysis to further verify the promoting effect of CPhGs on the expression of 3?-HSD in testis.At the same time,the CYP11A1 low expression cell model constructed with transfected siRNA in vitro was subsequently intervened with different concentrations of CPhGs solution(50,100,200?g/mL)to determine the level of CYP11A1 by WB.To verify whether CPhGs directly regulates CYP11 A1 to promote T biosynthesis.Results:1.Screening of anti-fatigue effect parts of C.tubulosaThe three different extraction parts of C.tubulosa have a certain improvement effect on the learning and memory ability of fatigued mice.Among them,the Oligose can significantly prolong the incubation period and reduce the number of errors in the dark avoidance experiment,shorten the time to complete the eight-arm maze and reduce the number of errors,make the levels of 5-HT,NE and E in the hypothalamus and cerebral cortex of central fatigue model mice lower than those in SD group,and the CRH,ACTH,CORT levels are close to normal level in blood circulation.At the same time,the Oligose can significantly prolong the swimming exhaustion time of exercise fatigue model mice,in which it can significantly reduce the accumulation of plasma metabolites LD,BUN,MDA.In addition,Oligose can significantly increase liver glycogen and muscle glycogen reserves.However,Glycan alone has a significant effect on improving sports fatigue indicators,and CPhGs has no obvious effect.Therefore,the above results suggest that the Oligose has the best effect on improving central and exercise fatigue in mice.2.Screening of promoting reproduction effect parts of C.tubulosaThe three different extraction parts of C.tubulosa have certain effects on the sexual behavior and blood biochemical indexes of reproductive injury model rats.CPhGs has the most significant effect.The specific performance is:the CPhGs can restore the plasma cAMP/cGMP value,which is one of the characteristics of kidney-yang deficiency,to significantly shorten the erection,capture latency,increase the number of captures.Increasing the wet weight of testis and seminal vesicles and the content of seminal plasma fructose and zinc.It can significantly increase the level of T in serum,and maintain the stability of FHS,GnRH,E2 and LH levels,and significantly improve the pathological morphology of testis.While Glycan only regulates FSH,LH and E2 levels,the effect of Oligose is not very obvious.Therefore,the above experimental results show that:CPhGs has the best effect on improving the reproductive ability of rats.3.Efficacy evaluation of different doses of CPhGs for promoting reproductionTwo dose levels of CPhGs(145,289 mg/kg)have certain effects on the erectile function,mating ability,organ wet weight,serum hormone LH and T levels,testicular pathological morphology changes in reproductive injury model mice.Low-dose CPhGs(72 mg/kg)have no obvious protective effect on reproductive damage caused by hydrocortisone.The high-dose CPhGs effectively shortened the erection,capture and ejaculation latency of model mice,and increased the number of captures and ejaculations.At the same time,high-dose CPhGs can increase the wet weight of the testes,epididymis,seminal vesicles and penis,and increase the levels of T and its upstream hormone LH,significantly improving the pathology of the testis.Positive drug TP can significantly improve the sexual behavior of reproductive injury model mice,but it increased the T level in the blood of mice sharply,far exceeding the normal level,and there was no ob'vious improvement effect on the reduction of LH level caused by hydrocortisone,nor could it alleviate the pathological damage state of the testis.The above experimental results suggest that CPhGs can improve the reproductive capacity of mice by increasing T level,and it is quantitatively dependent.The high-dose effect is the best,the main manifestation is a moderate increase in circulating T levels in reproductively injured mice,and its mechanism of action is different from that of TP.4.In vivo and in vitro mechanisms of CPhGs to increase T level and promote reproductionIn vivo experiments,WB results showed that the CPhGs can significantly increase the expression of steroid-producing enzymes CYP11A1 and dehydrogenase 3?-HSD in the T synthesis pathway in testis tissue,but it is not obvious effect for the transport protein StAR.As a positive control drug,TP inhibited the expression of CYP11A1.IF was further used for qualitative and quantitative detection of 3?-HSD expression.The results were consistent with those shown in WB.The expression of 3?-HSD was significantly increased after treatment with high-dose CPhGs.In vitro experiments,WB results showed that CPhGs(50?g/mL)can significantly increase the expression of CYP11A1 in normal cells.Compared with that,CPhGs were given after transfection of siRNA,CPhGs can still significantly increase the expression of CYP11A1,but the effect is somewhat reduced.The above results indicate that CPhGs can directly regulate the key enzyme CYP11A1 in the T synthesis pathway to promote the expression of its downstream 3?-HSD,thereby increasing T level.Conclusions:Screen and evaluate the efficacy of Oligose,Glycan and CPhGs in animal models for anti-fatigue and reproductive effects.The results showed that the Oligose can regulate the release of central neurotransmitters,maintain stable levels of blood hormones related to the hypothalamus-pituitary-adrenal axis,enhance liver and skeletal muscle glycogen storage capacity,reduce the accumulation of metabolites.Furthermore,it improves the learning,memory and exercise ability of the fatigue model mice.CPhGs can protect the testis from hydrocortisone damage and increase the wet weight of the testis and its accessory sex organs,maintain stable levels of blood hormones related to the hypothalamus-pituitary-gonad axis.It can significantly increase the expression of steroid-producing enzymes CYP11A1 and dehydrogenase 3?-HSD in the enzymatic reaction of T synthesis after cholesterol enters mitochondria through directly regulating the level of CYP11A1 in the T synthesis pathway,thereby promoting the biosynthesis of T and improving the reproductive ability of mice,and playing the role of tonifying kidney-yang.