Study On Microvascular Function Of Gynecological Cold Blood Stasis Syndrome And The Effect Of Wenjing Decoction Based On Rho/ROCK Pathway Mediated By ET-1

Part one Physiological Function of Ovary and Uterus in Rats with Gynecological Cold Coagulation and Blood Stasis Syndrome and the Effect of Wenjing DecoctionObjective and Significance:By observing the pathological changes and physiological functions of ovaries and uterus in rats with gynecological cold coagulation and blood stasis syndrome(CCBS),and to explore the effect of Wenjing Decoction.Methods:The rat model of gynecological CCBS syndrome was established by ice water bath method.The rats were divided into blank group,model group,BQ123 group,and Wenjing Tang(WJT)Decoction group.The general state of rats was observed and the estrous cycle was recorded.The levels of reproductive hormones T,E₂and P in serum were detected.The ovarian uterus index and HE staining were used to observe the pathological changes of ovary and uterus of rats.Results:Compared with the blank group,the rats in the model group showed symptoms and signs of cold blood stasis,such as shivering,less movement,hitting piles,slow reaction,dark ear,tongue,claw tail,dull coat,clear urine color,diarrhea,etc.The symptom integral was significantly increased(P<0.01),the estrus cycle was prolonged(P<0.01);T,E₂,P were reduced(P<0.01),the uterine index was significantly increased(P<0.01),but there was no statistical significance in ovary index.HE staining showed the number of ovarian follicles in the model rats was less,the diameter of blood vessels and the endometrium became thinner.Compared with the model group,the rats in WJT and BQ123 groups improved in varying degrees.The scores of symptoms decreased significantly(P<0.01),the estrous cycle shortened(P<0.01),E₂,P increased(P<0.01,P<0.05),the uterine index decreased(P<0.05),the number of ovarian follicles increased,and the diameter of blood vessels was normal.Conclusions:1.In addition to the general symptoms of cold coagulation and blood stasis syndrome,the model rats also have low ovarian and uterine functions,which are characterized by prolonged estrous cycle and decreased reproductive hormone levels,decreased follicle number in ovary and thinned endometrium.2.Wenjing Decoction can effectively improve the cold blood stasis and the hypofunction of ovary and uterus in model rats.3.ET-1 may be involved in the pathological mechanism of gynecological cold coagulation and blood stasis syndrome.Inhibition of ET-1 activity has therapeutic effect on gynecological cold coagulation and blood stasis syndrome.Part two Ovarian and Uterine microcirculation changees in rats with Gynecological CCBS syndrome of gynecology and the effect of Wenjing DecoctionObjectives and Significance:By observing the microcirculation and microvascular structure of rats with gynecological CCBS syndrome,the changes of microvascular function in the whole body and local ovarian and uterine tissues of rats with gynecological CCBS syndrome and the intervention effect of Wenjing Decoction were explored.Methods:The whole blood rheology,vasoconstriction factors in serum and ovarian tissues,microcirculation of auricle and uterus surface were monitored by speckle flow imaging system,CD34,the distribution and expression of and?-SMA,PCNA and SM22?protein were detected.Results:Compared with the blank group,the whole blood rheological indexes of the model group increased(P<0.01 or P<0.05),and also the expression of ET-1 in serum,NO decreased(P<0.01,P<0.01);while the imbalance of vasodilator-contractile factors in ovarian tissue of the model group,the microcirculatory blood flow of auricle and uterus of the model group decreased significantly(P<0.01),positive expression of antibody CD34decreased,and the protein of?-SMA,PCNA and SM22?decreased significantly(all P<0.01).The expression increased,mainly in ovarian and uterine vascular smooth muscle cells.Compared with model group,BQ123and Wenjing Decoction could significantly reduce ET-1 content in ovarian tissue(P<0.01,P<0.05),increase NO content(P<0.01,P<0.01),increase microcirculatory blood flow in auricle and uterus(P<0.01,P<0.01),increase microvessel density,decrease the protein expression of?-SMA(P<0.01,P<0.05),and SM22?(P<0.05,P<0.01),PCNA(P<0.01,P<0.01).Improved the damage status of local vascular endothelial cells and proliferation of vascular smooth muscle cells.Conclusions:1.There is microcirculation disturbance in rats with gynecological CCBS syndrome.The manifestations were abnormal blood flow and imbalance of vasodilator-contractile factors,decreased blood flow in local microcirculation.2.Vascular endothelial cell injury and contraction and proliferation of vascular smooth muscle cells in local ovarian and uterine tissues were also found.Microvascular contraction and remodeling was possible.3.Wenjing Decoction can improve the microcirculation of rats with gynecological CCBS syndrome.It can also restore microvessel status and relieve microvessel contraction and remodeling in local reproductive organs.Part three The mechanism of endothelin-mediated Rho/ROCK signaling pathway in rats with gynecological CCBS syndrome and the mechanism of Wenjing Decoction.Objectives and Significance:To explore the mechanism of endothelin-mediated Rho/ROCK signaling pathway in microvascular lesions of rats with gynecological CCBS syndrome and the mechanism of Wenjing Decoction,by detecting the expression of Rho A,ROCK1,MLCK m RNA and protein.Results:Compared with the blank group,the levels of ET-1,ET-AR,Rho A,ROCK1 and MLCK protein in the model group increased significantly(all P<0.01),while the contents of ET-BR and protein decreased significantly(P<0.01).Compared with the model group,the levels of ET-1,ET-AR,Rho A,ROCK1,MLCK m RNA and protein were significantly decreased in BQ123group and Wenjing Decoction group(all P<0.01),while the ET-BR increased(P<0.01,P<0.01).Conclusions:1.In the ovarian tissue of rats with gynecological cold coagulation and blood stasis syndrome,endothelin can activate Rho/ROCK signaling pathway by binding to downstream A receptor.2.The abnormal activation of Rho/ROCK signaling pathway mediated by endothelin may be involved in the mechanism of microangiopathy in rats with gynecological CCBS syndrome.3.Wenjing Decoction can improve the local microcirculation abnormality of reproductive organs in rats with gynecological CCBS syndrome by inhibiting excessive activation of Rho/ROCK signaling pathway,so as to achieve the goal of treating gynecological CCBS syndrome.Part four Effect of Wenjing Decoction on the Proliferation of Rat Thoracic Artery Smooth Muscle Cells Induced by Endothelin and Its MechanismsObjectives and Significance:To investigate the effects of serum containing Wenjing Decoction on the proliferation of rat thoracic artery smooth muscle cells induced by endothelin and the expression of Rho A,ROCK1 and MLCK,key factors of endothelin-induced Rho/ROCK signaling pathway.Methods:The effects of Wenjing Decoction containing serum,BQ123,Y-27632 on the proliferation of rat thoracic artery smooth muscle cells were detected by CCK8 method.The protein expressions of Rho A,ROCK1 and MLCK in VSMCs were detected by immunocytofluorescence and Western Blot methods.Results:The results showed that ET-1 of 10^-7mol/L could induce the proliferation of VSMCs,BQ123 of 10^-5mol/L or Y-27632 of 10^-6mol/L could significantly inhibit the proliferation of VSMCs induced by ET-1,and serum containing 10%Wenjing Decoction could significantly inhibit the proliferation of VSMCs induced by ET-1.Compared with the control group,the expression of Rho A,ROCK1,MLCK in ET-1-induced group was significantly increased.Compared with ET-1 induction group,the protein expression of Rho A,ROCK1 and MLCK in the serum group containing Wenjing Decoction was significantly decreased(all P<0.01).Conclusions:1.ET-1 can induce the proliferation of VSMCs.2.ET-AR inhibitor BQ123 and ROCK1 inhibitor Y-27632 could inhibit the proliferation of VSMCs induced by ET-1.It further suggests that Rho/ROCK signaling pathway may be involved in ET-1-induced VSMC proliferation.3.Wenjing Decoction can significantly inhibit the proliferation of rat thoracic artery smooth muscle cells induced by endothelin.The mechanism may be related to blocking the Rho/ROCK signaling pathway induced by ET-1.