The Mechanism Of Sodium Tanshinone ? A Sulfonate To Ameliorate Erectile Dysfunction In Hyperlipidemia Rats Through Antioxidant Stress

Erectile dysfunction(ED)is a clinically common male disease.ED development is more than a system of multiple factors,long-term,complex process of chronic,its pathogenesis is still unclear,so to explore the pathogenesis of ED,for looking for effective prevention and control measures,which is of great significance for individualized treatment.Corpus cavernosum smooth muscle(CCSM)plays an important role in the end of penile erection.Smooth muscle cells are divided into contraction and proliferative two types,the contraction are the main functions of the contraction and relaxation,maintain the nonnal morphological structure and function of organs,and proliferative mainly play a role in cell proliferation,migration.secretion and degradation of extracellular proteins.The study of phenotype is derived from vascular smooth muscle cell(VSMCs).and VSMCs is transformed from contractile type to synthetic and acquired proliferative capacity.This process is called phenotypic transformation.Contractile type contains the following indicators:a-actin(a-SMA),smooth muscle myosin heavy chain(SMMHC),basic protein calponinl(calponin1)and calmodulin(h-caldesmon).The synthetic marker is osteopontin(OPN).A large number of studies have shown that hyperlipidemia is an important factor in the development of ED,and hyperlipidemia can make corpora cavernosa endothelial cells and smooth muscle damage,causing dysfunction of the corpora cavernosa,but whether hyperlipidemia caused the phenotypic transformation of corpus cavernosum smooth muscle,the conversion from contractile to synthetic,resulting in smooth muscle dysfunction,has not been reported.Therefore,it is of great significance to study the phenotype transformation of the corpus cavernosum smooth muscle in the pathogenesis of hyperlipemia.The increase of oxidative stress caused by hyperlipidemia mediated ED is thought to be the main enzyme that produces ROS/superoxide in blood vessels.The increase of ROS production in hyperlipidemia can also affect the expression and activity of the key enzymes of L-Arg-NO-CGMP pathway,which can cause the L-Arg-NO-CGMP pathway to be impaired,resulting in impaired endothelial function.However,whether the oxidative stress mediated the corpus cavernosum smooth muscle damage and functional abnormalities,is through the L-Arg-NO-CGMP pathway in endothelial injury and indirectly affect the function of smooth muscle,or smooth muscle cells,the damage can be directly or endothelial and smooth muscle damage occurred at the same time together lead to ED,and the specific molecular mechanism is unclear.Oxidative stress damage,therefore,is likely to be key factors in the pathogenesis of ED,discussed oxidative stress damage on hyperlipidemia ED function of corpus cavernosum smooth muscle cell pathological changes and molecular mechanism of structural damage,both to elucidate the pathogenesis of ED and as an important target of clinical diagnosis and treatment of ED and prevent the happening of the ED has important significance.Tanshinone IIA Sodium Sulfonate(STS)is a traditional Chinese medicine extract of salvia miltiorrhiza,which has protective effect of heart and resistance to atherosclerosis,STS has many biological activities,including resistance to oxidative stress,diastolic blood vessels,anti-atherosclerosis?anti-inflammatory and anti-fat formation.STS is an effective antioxidant,which can break the chain reaction of peroxidation by scavenging lipid free radicals and increasing the activity of superoxide dismutase(SOD),which has multiple target effects.Clinically,we found that the treatment of ED patients also achieved better results with the improvement of cardiovascular disease in STS.Therefore,we hypothesized that the phenotypic transformation of the smooth muscle of the corpus cavernosum induced by oxidative stress caused by hyperlipidemia may play a key role in the pathological process of ED.Based on this assumption,this study will establish hyperlipidemia rat model to observe the changes of the cavernous smooth muscle cell phenotype and oxidative stress?then it will clear the role of oxidative stress damage on the process of ED and cavernous smooth muscle cells participate in cavernous fibrosis;On this basis,we further explore whether STS can ameliorate ED by anti-oxidative stress and improve the pathological changes of cavernous smooth muscle cells.It is believed that these studies will enrich ED's etiology and pathogenesis theory,and improve ED patients through anti-oxidative stress,which is expected to provide a new strategy for clinical diagnosis and treatment.Part 1 Phenotypic characteristics of CCSM cells and changes in oxidative stress levels in the tissue of the corpus cavernosum in hyperlipidemia ED ratsMethods:High fat diet to establish a rat model of hyperlipidemia,32 SD rats were randomly divided into two groups,respectively before and after modeling test in weight,total cholesterol,triglyceride,high density lipoprotein,low density lipoprotein,after modeling the use of cavernous nerve stimulation intracavernosal pressure measurement erectile function,simultaneous measurement of carotid artery pressure,the ratio was calculated.The morphological changes of tissue were detected by HE staining,and the intimal lipid deposition of the thoracic aorta was measured by oil red O staining,and the changes of CCSM content in rats were observed by Masson staining.QRT-PCR and western blotting methods related to the marker genes and proteins of the phenotype of smooth muscle in corpus cavernosum:Calponin,Myh11,OPN expressions as well as phenotype of smooth muscle cell regulatory factor Myocardin.Immunohistochemical detection of corpus cavernosum CollagenI,TGF-? expressions;measuring penile tissue SOD and GSH levels by chemoenzymatic methods.The oxidative stress state in ED rats with hyperlipidemia was preliminarily determined,and the correlation between phenotype transformation and ED in cavernous smooth muscle cells was observed.Results:the weight of the HR group was significantly increased compared with the NC group(P<0.001).According to carotid artery and corpus cavernosum pressure measurements,calculation of maximum corpora cavernosa stimulus pressure/carotid artery pressure ratio(intracavernous pressure/mean arterial pressure,ICP/MAP)ratio,the results showed that the difference of MAP have no statistical significance(P=0.282),but the difference of ICP was statistically significant(P<0.001)between HR and NC groups,ICP/MAP in NC group was significantly lower than HR group,the difference was statistically significant(P<0.05).The TC,TG and LDL significantly increased(P<0.001)but HDL decreased significantly(P<0.05)in HR&ED group.HE staining showed that liver cell structure was disorganized and a large number of vacuolar changes of lipid deposition in HR&ED rats.Oil red O staining showed that lipid deposition occurred in the inner and outer layers of the aorta in HR&ED rats.Compared with the NC group,the expressions of Calponin,Myh11 and Myocardin mRNA was significantly decreased(P<0.05,P<0.05,P<0.001),while OPN significantly increased(P<0.001)in the HR&ED groups.Western blot(WB)confirmed that Myocardin,Calponin and Myh11 protein were significantly decreased(P<0.05).while OPN expression increased(P<0.05)in the HR&ED groups.Immunohistochemistry showed that the expressions of Collagenland TGF-?significantly increased in HR&ED group(P<0.05),suggesting that the increased Collagen deposition and fibrosis in corpora cavernosa in HR&ED rats.Masson staining showed that the number of CCSM in the HR&ED group was increased,but the comparison with the NC group was not statistically significant(P<0.05).The content of SOD and GSH in the penis of HR&ED rats was significantly lower than that in the NC group(P<0.05).Part 2 The effect of Acetylcysteine(NAC)on erectile function of hyperlipidemia ED ratsMethods:36 male SD rats were randomly divided into 4 groups:the normal group(NC).hyperlipidemia group(HR),hyperlipidemia+saline group(HR+NS)and hyperlipidemia+acetylcysteine group(HR+NAC).After the model was made,the pressure was measured in the penis.Masson staining evaluation content of corpus cavernosum smooth muscle;Chemical enzymatic detection rat corpus cavernosum tissue SOD,GSH,MDA and the level of Testosterone;Corpus cavernosum tissue Collagenl,TGF?,HO-1 expression and smooth muscle phenotype transformation related factor a-SMA,SMMHC,Calponin,Myocardin and OPN expressions were measured by immunohistochemistry,QRT-PCR and western blotting methods,which preliminarily discussed the effect of acetylcysteine(NAC)in rat corpus cavernosum smooth muscle cell phenotypic transformation.Results:ICP/MAP shows:the difference of MAP have no statistical significance among groups(P=0.403);the difference of ICP have statistical significance among groups(P<0.001),ICP/MAP in HR group have statistical significance compared with NC group,HR+NS group and HR+NAC(P<0.001,P=0.248,P<0.001).Compared with the HR group,the TG in the HR+NAC group significantly decreased(P<0.001)and HDL increased significantly(P<0.001).HE staining showed a disorder of liver cells in hyperlipidemia rats and a large number of vacuolar changes of lipid deposits.Oil red O staining results showed that the lipid deposition in middle membrane layer and outer membrane as well as following with some intimal thickening in rat aorta was seen in the HR and HR+NS group,but HR+NAC group rat aorta layers not seen obvious lipid;The content of SOD and GSH in the penis sponge in the HR+NAC group was higher and the MDA content decreased(P<0.05)than that in the HR group(P<0.05).Compared with the HR group,Myocardin,SMMHC mRNA significantly increased in the HR+NAC group(P<0.001),and OPN significantly decreased(P<0.001),but ?-SMA has no statistically significant difference between the two groups(P=1.000).Compared with the HR group and HR+NS group,the expression of SMMHC,Calponin and Myocardin protein was increased in the HR+NAC group(P<0.05),while the OPN expression decreased(P<0.05),but a-SMA has no statistically significant difference between the two groups(P=0.699).Immunohistochemistry showed that the expression of HO-1 in the HR+NAC group increased significantly(P<0.05)and CollegeIand TGF-? decreased compared with the HR group(P<0.05).Masson staining showed that compared with the HR+NAC group,the number of CCSM in the HR group and HR+NS group was increased,but the differences among the groups were not statistically significant(P=0.243).There was no statistically significant difference between serum testosterone in the NC group,HR group,HR+NS group and HR+NAC group(P=0.081).Part 3 The mechanism of sodium tanshinone ? A sulfonate to improve erectile dysfunction in hyperlipidemia rats through antioxidant stressMethods:60 SD males were randomly divided into 5 groups:NC,HR,H R+NS,HR+NAC,hyperlipidemia+tanshinone ? A sulfonate group(HR+STS),and under the use of cavernous nerve stimulation evaluation corpus ca vernosum pressure.Phenotypic regulation factor:Myocardin and smooth muscle phenotypic markers factors of a-SMA,SMMHC,Calponin OPN,heme oxygen ase-1(HO-1),as well as the nuclear factor E2 related transcription factor 2(N RF2)were measured by QRT-PCR and Western blotting in corpus cavernosum tissue.The changes of CCSM content in penile were observed by Masson stai ning.Immunohistochemical detection of CollagenI,TGF-?,HO-1 and eNOS exp ressions.Chemical enzymatic detection of SOD,GSH and MDA levels in seru m and penile cavernous tissue.In order to explore the mechanism of STS anti oxidation to improve penile erectile function in hyperlipidemia rats.Result:MAP has not statistically significant difference among the groups(P=0.202);ICP and ICP/MAP among the groups was statistically significant diffe rence(P<0.001);The NR group has statistically significant difference compared with the NC group,HR+NAC group and HR+STS group;There was no signif icant difference between HR+NAC group and HR+STS group(P=0.253).Comp ared with the HR group,the TG level of the HR+STS group was significantly reduced(P<0.05).Compared with the HR group,the TG level of the HR+ST S group was significantly reduced(P<0.05).The aortic oil red O staining sho wed no significant lipid deposition in the thoracic aorta of the HR+STS group The content of SOD and GSH in the serum and cavernous tissue of the peni s of HR+STS group was higher than that in the HR group(P=0.004 and P=0939,P=0.003 and P=0.014),while MDA content decreased(P=0.004 and P=0.001).Compared with HR group and HR+NS group.a-SMA,SMMHC,Myocard in mRNA expressions significantly increased in HR+STS group,while OPN ex pression was decreased(P<0.05).Compared with the HR group,the expressions of a-SMA,SMMHC,Calponin and myocardin was significantly increased in t he HR+STS group(P<0.05),while the OPN expression was decreased(P<0.05);NRF2 and HO-1 were significantly higher in HR+NAC group and HR+STS g roup(P<0.05).Compared with the HR group and the HR+NS group,Collegela nd TGF? were reduced(P<0.05),but the expressions of HO-1 and eNOS were increased(P<0.05)in the HR+STS group.Masson staining showed that there was no statistically significant difference among groups(P=0.176).Serum testos terone levels in rats showed no statistically significant difference between grou ps(P=0.491).Conclusion:1.The model of ED rats with hyperlipidemia was successfully established and the oxidative stress level in the body was increased.2.The model of hyperlipidemia ED rats resulted in the phenotype transformati on of the corpus cavernosum and penile tissue fibrosis;The proliferation p henotype of CCSM in hyperlipidemia rats was related to the occurrence of ED.3.NAC could improve the anti-oxidative stress level of HR&ED rat serum an d penile cavernous tissues,inhibiting the fibrosis process and maintaining t he contractile phenotype of CCSM.4.STS could ameliorate erectile dysfunction of hyperlipidemia ED rat,improv ing anti-oxidative stress level,keeping the corpora cavernosa CCSM contrac tility phenotype and protecting endothelial function,which mechanism may be through the NRF2/HO-1 signaling pathways remaining CCSM cell pheno type5.STS could reduce hyperlipidemia ED rats triglyceride levels,but was not a ffect their testosterone levels.