| BackgroundIn the traditional Chinese medicine theory,the kidney is the main reproductive agent and is closely related to the“renal essence”.Insufficient kidney essence leads to lack of reproductive essence,which leads to infertility.The material basis of“kidney essence”is still unknown.Our research team put forward the hypothesis that“Erythropoietin?Erythropoietin,EPO?may be an important biological material basis for kidney essence”,and preliminary verified the“renal essence in the brain”,“kidney essence in the bone”,“kidney essence boosts immunityand”and other aspects of relevance to EPO.In this study,we observed the changes of EPO in three male animal models?EPO gene knockout models and two models of reproductive dysfunction due to kidney deficiency?,and the improvement of three male animal models by supplementing exogenous EPO or giving the classic formula for nourish the kidney and strengthen the essence.The role and mechanism are to reveal the effects of EPO on the reproductive function of males with kidney deficiency.Therefore,from the perspective of reproduction,it is proved that EPO may be an important material basis of“Kidney essence”,and explain the traditional Chinese medicine theory of“kidney governing reproduction.”This research was supported by the National Natural Science Foundation of China?81473549?and the2018 Central University Basic Research Business Fee Student Program?XDJK2018D027?.Purpose1.Observe the changes of EPO in male EPO knockout mice,naturally aging mice,and rats with reproductive hypoplasia of kidney deficiency,and the reversal effect of exogenous rhEPO;2.To study the improvement effect of"nourish the kidney and strengthen the essence"classic recipe You-Gui-Yin on EPO gene knockout male mice,male natural aging mice,and kidney deficient reproduction rats,and to explore whether the mechanism is related to HIF1?-STAT5 pathway;3.The effects of EPO on the reproductive function of males with kidney deficiency are to be revealed through the above test.From the perspective of reproduction,it was proved that“EPO may be an important material basis of“kidney essence””,and explained the Chinese medicine theory of“Kidney essence stores the essence of reproduction”.Methods and results1.Correlation between EPO and reproductive dysfunction in males with kidney deficiencyMethods:?1?Establish systemic conditional knockout mice for EPO genes:In this study,"target gene-anchored EPO?flox/flox?mice"and"knockout tool UBC-CreERT2 mice"provided by genecorporation for mating,identification,and screening male“EPO?flox/flox?-CreERT2RT2 mice”.EPO?flox/flox?-CreERT2 mice?EPO knockout mice?and EPO?flox/flox?mice?Wild Type mice?were selected and raised to 8 weeks of age.Then select8-week-old male EPO?flox/flox?-CreERT2 daughter mice?to be EPO gene knockout mice?and male EPO?flox/flox? mice?wild type,WT?,and inject 100 mg·kg-1 Tamoxifen?drug that performs EPO gene knockout?for 5 consecutive days,from the 6 th day on,the mice are normally fed for 28 days,the body temperature and body weight of the mice are measured every 7 days,and the mice are sacrificed on the 34th day Check various indicators.Among them,EPO?flox/flox?-CreERT2 mice became"EPO-conditional systemic knockout?EPO-KO?mice"after tamoxifen injection.The expression of EPO protein in the kidney and testis of the mice was detected to calculate EPO The knockout rate of genes in the above tissues to determine whether the knockout was successful.?2?A mouse model of male reproductive dysfunction due to natural aging caused by kidney deficiency was reproduced.The mice were normally raised for 12 months to make a model.The model was successfully judged as follows:?1?Compared with the young mice,each aging model mouse has significantly reduced autonomous activity,new object recognition,and stick rotation time.The serum SOD and T-AOC are reduced,and the increase in MDA is the success of the aging model;?2?Compared with the average of the young mice,the serum UREA and CREA in each mouse increased,and ACTH,T,T3,or T4 decreased in each mouse;?3?Compared with the mean of the young mice in the control group,the T,E2,LDH,and DHT in the serum of each mouse decreased,the number of sperm decreased,and the rate of abnormal sperm abnormality increased,which was a successful model of reproductive impairment.?4?Meeting the above?1??2??3??3?at the same time is the success of the model of male dysfunction due to kidney deficiency in naturally aging mice.The mice with successful model of natural aging kidney deficiency with male reproductive dysfunction were used for follow-up experiments.?3?The adenine-induced male model of hypogonadism in rats with renal deficiency was reproduced.The SD rats were randomly divided into normal groups and models.The models were administered with adenine 150 mg·kg-1 for 14days.The model's success criteria were:?1?Compared with the normal group rats,the UREA and CREA levels in each rat's serum increased,and the ACTH,T,T3,or T4 levels decreased after the model was established.?2?Compared with normal rats,the T,E2,LDH,DHT,and FSH in the serum of each rat were reduced,the sperm deformity rate was increased,and the number was decreased.Sexual behavior:the number of rides,the number of insertions,and the number of ejaculations are reduced,which means that the reproductive function impairment model is successful.?3?At the same time meeting the above?1??2?The two judgment criteria are the success of the kidney deficiency reproductive function model.Rats with successful kidney deficiency with male reproductive dysfunction models were selected for follow-up experiments.The appearance and morphology,blood routine,serum biochemical parameters,organ pathology,sperm count,sperm abnormality rate,and key protein content of HIF1?-STAT5 pathway in kidney and testis were examined.Results:?1?"EPO gene conditional systemic knockout mice"has been successfully established,and the male mice have the clinical manifestations of deficiency of kidney essence and reproductive dysfunction.The knockout rates of EPO gene in kidney and testis of EPO knockout mice were 77.53%and 76.86%,respectively.Compared with the wild-type mice:the animal's hair color becomes worse,appear ascites,body weight,body temperature and food intake are significantly reduced;liver,spleen,lung,kidney,pituitary,testis,epididymis,prostate,seminal vesicle,spine tissue morphology are significantly changed;Sperm count decreased significantly,sperm abnormality rate increased significantly,sperm appearance and morphology were significantly changed;serum EPO decreased significantly,SEPOR increased significantly,the ratio of EPO to SEPOR significantly reduced;blood routine indicators Gran%,Gran,MID,MID%,RBC,HCT significantly decreased,HGB,LY,MCH,WBC significantly increased;renal function indicators UREA,CREA significantly increased;thyroid indicators ACTH,CORT,T3,T4 significantly reduced;oxidative stress indicators MDA significantly increased,SOD,GSH-Px,T-AOC and LPO were significantly reduced;immune indicators C3,C4,IL-2,IL-6,IGA,IGG,IGMwere significantly reduced;reproductive indicators T,E2,LH,DHT,FSH were significantly reduced;HIF1?-in kidney and testis STAT5 pathway protein decreased significantly?all p<0.05?.?2?The mouse model of reproductive dysfunction due to natural aging caused by male kidney deficiency was successfully established,EPO was significantly reduced,and related pathway proteins were significantly abnormal.Compared with young mice,aging model mice:poor appearance and hair color,lower body temperature and body weight;glomerular vacuoles,more inflammatory factors;incomplete testicular body shape,atrophy and congestion of seminiferous tubules;sperm count significantly decreased,the sperm abnormality rate increased significantly;blood routine indexs RBC,HCT,HGB,MID,MID%,MCH,PCT significantly decreased;serum UREA,CREA significantly increased;ACTH,CORT,T3,T4significantly reduced;MDA,LPO significantly reduced Increased,SOD,GSH-PX,T-AOC decreased significantly;T,E2,LH,DHT,FSH all significantly decreased;the times of independent activity and time of turn stick significantly decreased,the identification index increased significantly;serum EPO significantly reduced,SEPOR significantly increased,the ratio of EPO to SEPOR significantly reduced;kidney and testis HIF-1?,EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?protein expressions were significantly reduced?all p<0.05?.?3?The model of adenine-induced renal deficiency in male reproductive dysfunction was successfully established and EPO was significantly decreased,and HIF1?-STAT5pathway protein was significantly abnormal.Compared with the normal group,the rats in the model group:poor appearance and hair color,lower body weight and body temperature;the kidneys became whiter,the glomerular cavity was larger,adenine crystals in the tubules,coagulative necrosis,and inflammatory cells More infiltration;decreased blue-stained calcium salt deposits in the lumen of the testicular body,atrophy of the seminiferous tubules,loosening of the seminiferous tubules,thickening and hyperemia of the blood vessel wall;sperm appearance and internal morphological changes;significantly reduced sperm counts,sperm abnormality rate increased significantly;blood routine indexs Gran%,Gran,MID,MID%,RBC,HCT,PCT,MCH decreased significantly,LY,WBC increased significantly;serum UREA,CREA increased significantly;ACTH,CORT,T3,T4 content Significantly reduced;T,E2,LH, DHT,FSH were significantly reduced;mount frequency,intromission frequency and ejaeulation frequency were significantly reduced;serum EPO was significantly reduced,SEPOR was significantly increased,the ratio of EPO to SEPOR significantly reduced;kidney and testis HIF-1?,EPO protein expression was significantly reduced Decreased,EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?protein expression increased significantly?all p<0.05?.2.Observation on the effect of exogenous rhEPO on the reproductive function and EPO signal pathway in males with low kidney functionMethods:?1?Reversal of exogenous rhEPO on EPO knockout male mice:Take8-week-old male EPO knockout mice and the wild-type mice,divided into 4 groups:EPO-KO group,WT group,EPO-KO+rhEPO 500 IU·kg-1,WT+rhEPO 500 IU·kg-1,5 animals in each group.Each group of rhEPO was injected subcutaneously with rhEPO every three days.The EPO-KO group and the WT group were given an equal volume of normal saline for 28 days.Animal appearance,body weight,body temperature,behavior,blood routine,serum biochemical indicators,organ pathology,sperm morphology,sperm number,sperm abnormal rate,Contents of key proteins of HIF1?-STAT5pathway in kidney and testis were measured.?2?Reversal test of exogenous rhEPO on mice with natural aging and kidney deficiency:Natural aging model mice are divided into 4 groups:model group,rhEPO 250 IU·kg-1,500 IU·kg-1,1000 IU·kg-1,15 mice per group,2-month-old male mice were used as the young control group.Each group of rhEPO was subcutaneously injected with a corresponding dose of rhEPO every three days,and the aging model group was given an equal volume of normal saline for 28days.Animal appearance,body weight,body temperature,behavior,blood routine,serum biochemical indicators,organ pathology,sperm morphology,sperm number,sperm abnormal rate,Contents of key proteins of HIF1?-STAT5 pathway in kidney and testis were measured.?3?Reversal test of exogenous rhEPO on adenine-induced male reproductive dysfunction in rats with kidney deficiency:The model rats were randomly divided into 4 groups:model group,rhEPO 500 IU·kg-1,1000 IU·kg-1,1500IU·kg-1 15 rats in each group,the balance test between the groups,no significant difference after administration,another 15 rats in the normal group.Starting on the 15th day,except for the normal group,the model was maintained at 150 mg·kg-1 adenine every other day,and rhEPO was injected subcutaneously every three days,last 3 days.Animal appearance,body weight,body temperature,behavior,blood routine,serum biochemical indicators,organ pathology,sperm morphology,sperm number,sperm abnormal rate,Contents of key proteins of HIF1?-STAT5 pathway in kidney and testis were measured.?4?Protective effect of exogenous rhEPO on three TM4 cell injury models and its mechanism:TM4 cells were used as a model,and three kinds of interventions were performed:the EPO gene was knocked out using Crispr Cas9technology;JAK2 and STAT5 protein inhibitor treatmen;hypoxia and hypoglycemia damage.After cell intervention,the cell proliferation,LDH leakage rate,mitochondrial membrane potential,and effects on HIF1?-STAT5 pathway were observed after administration of rhEPO 12.5 U·mL-1.Results:?1?Exogenous rhEPO significantly improves the physique and reproductive function of males with kidney Deficiency and reproductive dysfunction:?1?Exogenous rhEPO significantly improves EPO gene knockout male mice's physique and reproductive function.Compared with the EPO gene knockout group,the exogenous rhEPO 500 IU·kg-11 group:better appearance,hair color,and increased body temperature and body weight;full glomeruli,fewer vacuoles,and fewer inflammatory factors;more complete testicular body shape,curved Seminiferous tube atrophy and less hyperemia;sperm count increased significantly,sperm deformity rate decreased significantly;blood routine Gran%,Gran,MID,MID%,RBC,HCT significantly increased,HGB,LY,MCH,WBC significantly decreased;serum UREA,CREA significantly decreased;ACTH,CORT,T3,T4 significantly increased;MDA significantly decreased,SOD,GSH-PX,T-AOC,LPO significantly increased;T,E2,LH,DHT,FSH significantly increased;small The number of mouse autonomous activities,the time of turning the rod increased significantly,and the new object recognition index decreased significantly?all p<0.05?.?2?Exogenous rhEPO significantly improves the physical and reproductive functions of male mice with natural aging and kidney deficiency.Compared with the aging model group,rhEPO250,500,1000 IU·kg-1 groups'mice have better appearance,hair color,elevated body weight and body temperature;full glomeruli,fewer vacuoles,and fewer inflammatory factors;more complete testicular body morphology,and seminiferous tubules Atrophy and less hyperemia;Sperm count increased significantly,sperm abnormality rate decreased significantly;RBC,HCT,HGB,MID,MID%,MCH,PCT of blood routines increased significantly;UREA and CREA of serum decreased significantly;ACTH,CORT,T3,T4 increased significantly;MDA and LPO decreased significantly,SOD,GSH-PX,and T-AOC increased significantly;T,E2,LH,DHT,and FSH increased significantly;the number of autonomous activities of mice increased significantly,the times of independent activity and time of turn stick significantly increased,the identification index significantly reduced?all p<0.05?;?3?Exogenous rhEPO significantly improves constitution and reproductive function of adenine-induced male reproductive dysfunction in rats with kidney deficiency.Compared with the model group,rhEPO 500,1000,1500 IU·kg-1 groups'rats have better appearance,hair color,weight,and body temperature;kidney appearance and morphology are significantly improved,the glomerular cavity becomes smaller,the adenine crystals in the renal tubule become less,necrosis reduced,inflammatory cell infiltration reduced;morphology of testicular bodies tended to be normal,blue-stained calcium salts in the lumen were reduced,atrophy of the seminiferous seminiferous tubules was reduced,vasodilatation of the seminiferous seminiferous tubules became thicker,blood vessel walls became thicker,and hyperemia was reduced sperm morphology and tissue structure lesions were significantly alleviated;sperm counts increased significantly and sperm abnormality rate decreased significantly;blood routine Gran%,Gran,MID,MID%,RBC,HCT,PCT,MCH increased significantly,LY,WBC decreased significantly;serum UREA and CREA were significantly reduced;ACTH,CORT,T3,and T4 contents were significantly increased;T,E2,LH,DHT,and FSH were significantly increased;the number of rides,mount frequency,intromission frequency and ejaeulation frequency significantly increased?all p<0.05?.?2?Exogenous rhEPO significantly regulates EPO signaling pathway in males with kidney deficiency and reproductive dysfunction:?1?Exogenous rhEPO significantly increased EPO content and expression of signaling pathway proteins in EPO knockout male mice.Compared with EPO knockout mice,rhEPO 500 IU·kg-1 group mice,serum EPO increased significantly,SEPOR decreased significantly,the ratio of EPO to SEPOR increased significantly;EPO,p-EPOR?Tyr368??p-JAK2?Tyr570??p-STAT5?Ser725?protein of kidney and testis expression increased?all p<0.01?;?2?Exogenous rhEPO significantly increases EPO content and expression of signaling pathway proteins in naturally aging kidney-deficient male mice.Compared with the natural aging group,rhEPO 500 IU·kg-1 group mice,serum EPO increased significantly,SEPOR decreased significantly,the ratio of EPO to SEPOR increased significantly;expression of HIF-1?,EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?proteins increased?all p<0.01?;?3?Exogenous rhEPO regulates the expression of EPO and its signaling pathway proteins in adenine-induced male reproductive failure rats with kidney deficiency.Compared with the model group,rhEPO 250 IU·kg-1,500 IU·kg-1 groups'serum EPO significantly increased,rhEPO1500 IU·kg-1 group significantly decreased EPO,rhEPO groups significantly decreased SEPOR,the ratio of EPO and SEPOR.HIF-1?and EPO protein increased,and the expression of EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?protein decreased in kidney and testis?all p<0.05?.?3?Exogenous rhEPO has a significant protective effect on TM4 cells damaged by EPO gene knockout or hypoxia,but has no improvement on TM4 cells inhibited by EPO downstream pathways.?1?Exogenous rhEPO has significant protective effect on TM4 cells damaged by EPO gene knockout.Compared with the model group,the rhEPO 12.5 IU·mL-1 significantly promoted TM4 cell proliferation;significantly inhibited the changes in LDH leakage and mitochondrial membrane potential caused by model cell damage;caused EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?protein expression increased?all p<0.05?.?2?Exogenous rhEPO has no significant protective effect on TM4 cells inhibited by JAK2 and STAT5 proteins.Compared with the model group,rhEPO 12.5IU·mL-1 no significant changes in TM4 cell proliferation,LDH leakage rate,mitochondrial membrane potential and HIF-1?,EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?.?3?Exogenous rhEPO has a significant protective effect on TM4 cells damaged by hypoxia and hypoglycemia.Compared with the model group,the rhEPO 12.5 IU·mL-1significantly promotes TM4 cell proliferation;significantly inhibits LDH leakage rate and changes in mitochondrial membrane potential;makes HIF1?,EPO,EPO,p-EPOR?Tyr368?,p-JAK2(Tyr570,p-STAT5?Ser725?protein expression increased?all p<0.05?.It is suggested that EPO and its signals pathway have important regulatory effects on male reproductive function cells.3.Improvement of YGY on Kidney Deficiency Male Reproductive Animals and Its Regulation of EPO Signal PathwayMethods:?1?YGY test on EPO knockout male mice:Take 8-week-old male EPO knockout mice and the wild-type mice,divided into 4 groups:EPO-KO group,WT group,EPO-KO+YGY 10 g·kg-1,WT+YGY 10 g·kg-1,5 in each group.Each group of YGY was given the corresponding dose of YGY by gavage.The EPO-KO group and WT group were given the same volume of normal saline daily for 28 days.Detection methods and indicators are the same as"Method 2?1?".?2?Test of YGY on male mice with natural aging and kidney deficiency due to natural aging:Natural aging model mice are divided into 4 groups:aging model group,YGY 10 g·kg-1,20 g·kg-1 In the group of 1,40 g·kg-1,15 rats in each group,15 male C57BL/6J mice of 2 months old were the young control group.Each group of YGY was given an appropriate daily dose of YGY,and the aging model group was given an equal volume of normal saline for 28days.Detection methods and indicators are the same as"Method 2?2?".?3?Test of YGY on adenine-induced male reproductive dysfunction in rats with kidney deficiency:Rats with successful adenine modeling were randomly divided into 5groups:model group,YGY 10 g·kg-1,20 g·kg-1,20 g·kg-1 group,positive control group(Guilu Erxian cream 10 g·kg-1 group),after there was no significant difference in the balance test between the groups for administration,15 normal rats were taken as the normal group.Beginning on the 15th day,in addition to the normal group,each group was given a 150 mg·kg-1 adenine gavage maintenance model every other day.YGY each group and the GL group were given gavage once a day,last 46 days.Detection methods and indicators are the same as"Method 2?3?".?4?Protective effect and mechanism of YGY on TM4 cells damaged by hypoxia and hypoglycemia:The intervention method is the same as"Method 2?4?”.After cell intervention,the effects of cell proliferation,LDH leakage rate,mitochondrial membrane potential,and HIF1?-STAT5 pathway after administering YGY 100 ug·mL-1 were investigated.Results:?1?YGY significantly improves the physique and reproductive ability of males with kidney deficiency and reproductive dysfunction.?1?YGY did not significantly improve the physical fitness of EPO knockout male mice.YGY 20g·kg-11 group showed no improvement in appearance,coat color,body weight and body temperature;no improvement in pathology of kidneys and testes;no improvement in sperm count and sperm abnormality rate;blood routine indexs MCH,LY,RBC,HCT,PCT,Gran,Gran%,Mid,Mid%were not significantly changed,WBC,HGB,LY%were significantly reduced?all p<0.05?;serum UREA,CREA,ACTH,CORT,T3,T4,MDA,LPO,SOD,GSH-PX,T-AOC,T,E2,DHT,FSH were not significantly changed,LH was significantly increased?P<0.05?;the times of independent activity and time of turn stick not obvious changed,the identification index significantly reduced?all p<0.05?.?2?YGY significantly improved the physical fitness and reproductive ability of mice with natural aging and kidney deficiency.Compared with the aging model group,mice in YGY 10 g·kg-1?20 g·kg-1?40 g·kg-1 group have better appearance,hair color,elevated body weight and body temperature;full glomeruli,fewer vacuoles,and fewer inflammatory factors;more complete testicular body shape,curved seminal duct atrophy and less hyperemia;sperm count increased significantly,sperm abnormality rate decreased significantly;blood routine indexs RBC,HCT,HGB,MID,MID%,MCH,PCT decreased significantly;serum UREA,CREA decreased significantly;ACTH,CORT,T3 And T4 were significantly increased;MDA and LPO were significantly reduced,SOD,GSH-PX,and T-AOC were significantly increased;T,E2,LH,DHT,and FSH were significantly increased;the number of autonomous activities of mice and the time of rod rotation increased significantly;the times of independent activity and time of turn stick significantly increased,the identification index significantly reduced?all p<0.05?.?3?YGY significantly improves physical and reproductive performance of adenine-induced kidney deficiency male reproductive function in rats.Compared with the model group,rats in YGY 10 g·kg-1?20 g·kg-1?40 g·kg-11 groups have better appearance,hair color,weight,body weight and body temperature.The appearance of kidneys has improved significantly,the glomerular cavity becomes smaller,and the adenine crystals in the renal tubules become less.Decreased coagulative necrosis and decreased inflammatory cell infiltration;morphology of testicular bodies tended to be normal,blue-stained calcium salts in the lumen decreased,vasospermia atrophy was reduced,vasospermia was loosened,blood vessel walls thickened,and congested the condition was alleviated;sperm morphology and tissue structure lesions were significantly relieved;sperm counts increased significantly,and sperm abnormality rates decreased significantly;blood routine indexs Gran%,Gran,MID,MID%,RBC,HCT,PCT,MCH increased significantly,and LY and WBC decreased significantly;Serum UREA and CREA were significantly reduced;ACTH,CORT,T3,and T4 levels were significantly increased;T,E2,LH,DHT,and FSH were significantly increased;sexual behavior:the number of rides,mount frequency,intromission frequency and ejaeulation frequency significantly increased?all p<0.05?.?2?YGY significantly regulates EPO and its signaling pathway in males with kidney deficiency and reproductive dysfunction.?1?YGY has no significant effect on regulating EPO content and protein expression of signaling pathway in EPO gene knockout male mice.Compared with EPO knockout mice,there was no significant change in serum EPO and SEPOR in the YGY 20 g·kg-1 group of mice;There was no significant change in the content of HIF-1?,EPO protein,EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?protein in kidney and testis.?2?YGY significantly increased the expression of EPO and its signaling pathway proteins in male mice with natural aging and kidney deficiency.Compared with mice in the natural aging group,mice in10 g·kg-1?20 g·kg-1?40 g·kg-1 groups of YGY,serum EPO significantly increased,SEPOR decreased significantly,serum EPO and the ratio of EPO to SEPOR significantly increased;Increased expression of EPO and HIF1?protein and decreased expression of p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?protein in kidney and testis?all p<0.01?.?3?YGY significantly increased the expression of EPO and its signaling pathway proteins in adenine-induced reproductive male dysfunction in rats with kidney deficiency.Compared with the model group,the serum EPO and ratio of EPO to SEPOR in10 g·kg-1?20 g·kg-1?40g·kg-1 groups of YGY significantly increased,the SEPOR decreased significantly;the expression of EPO,p-EPOR?Tyr368?protein in kidney and testis increased,p-JAK2?Tyr570?,p-STAT5?Ser725?protein expression decreased?all p<0.05?;?3?YGY has a significant protective effect on TM4 cells damaged by hypoxia and hypoglycemia,but has no effect on TM4 cells with EPO gene knockout or EPO downstream signaling pathway blocked.?1?YGY did not significantly improve TM4 cells damaged by EPO gene knockout.Compared with the model group,YGY100 ug·mL-1 had no significantly changed of TM4 cell proliferation,mitochondrial membrane potential,HIF-1?,EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5proteins.?2?YGY has no significant improvement on TM4 inhibited by JAK2 and STAT5 protein.Compared with the model group,YGY 100 ug·mL-1 had no significant changes in TM4 cell proliferation,mitochondrial membrane potential,and HIF-1?,EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?proteins.?3?YGY significantly protects TM4 cells from hypoxia and hypoglycemia.Compared with the model group,YGY 100 ug·mL-11 significantly promoted TM4 cell proliferation;significantly inhibited the LDH leakage rate and changes in mitochondrial membrane potential caused by model cell damage;caused HIF-1?,EPO,p-EPOR?Tyr368?,p-JAK2?Tyr570?,p-STAT5?Ser725?protein expression increased?all p<0.05?.It is suggested that the protective effect of YGY on male germ cells depends on EPO and its signaling pathway.Conclusion1.Loss or reduction of EPO in vivo is closely related to kidney deficiency constitution and male reproductive dysfunction in three experimental animals:EPO gene knockout model,natural aging model,and adenine model;2.Supplementation of exogenous rhEPO has significant reversal effects on kidney deficiency constitution and low male reproductive function in EPO gene knockout model,natural aging model and adenine model;3.Invigorating the essence of classical prescriptions YGY has significant effects on improving the physique and reproductive function of two kinds of males with male reproductive dysfunction:natural aging model and adenine model.Its role is closely related to the increase in protein expression of EPO and its downstream signaling pathway HIF-1?-STAT5.But no significant improvement on EPO knockout male mouse models;4.Intervention tests of three TM4 cell models of exogenous rhEPO and YGY on EPO gene knockout,JAK2 and STAT5 protein inhibition,and hypoxia and glucose damage showed that EPO and its downstream signaling pathways are the key links to protect cell damage,it is also a key link for YGY to play a role;5.In this paper,a mouse model of conditional systemic knockout of EPO gene was established,which proved the similarity between the phenotype of male animals with EPO gene deletion and the clinical manifestations of patients with"deficiency of kidney essence"and supported the hypothesis of"EPO may be an important biological material basis for kidney essence";6.This article reports the effects of EPO on the male reproductive function of kidney-deficient animals,validates the hypothesis that"EPO may be an important material basis for'nephrine essence'"from the perspective of reproduction,and explains the traditional Chinese medicine theory of'refining essence of kidney essence'. |
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