The Role Of RhoA/ROCK Pathway In Estrogen-induced Rat Colonic Relaxation

Background Chronic constipation(CC)is one of the most common gastrointestinal motility disorders in the world,which seriously affects people's physical and mental health and quality of life.Recent data show that 8.2% of the population in China is plagued by constipation(about 100 million people).The ratio of males and females is about 1:2.3.The incidence rate in women of childbearing age and pregnancy is higher.Animal experiments have found that estrogen can inhibit colonic movement in rats and cause constipation.Phosphorylation of myosin light chain(MLC)is one of the key factors in smooth muscle contraction.The level of MLC phosphorylation is regulated by calcium/calmodulin-dependent myosin light chain kinase(MLCK)and calcium-independent myosin light chain phosphatase(MLCP).Evidence has shown that RhoA/ROCK pathway is the main way to regulate MLCP(Ca2+ sensitization).ROCK inhibits MLCP activity through two pathways: One is the ability to phosphorylate MYPT1,the regulatory subunit of MLCP,so that MLC dephosphorylation cannot be catalyzed,then the level of phosphorylated MLC in the cytoplasm increases,and the smooth muscle contraction.The second is to phosphorylate CPI-17,causing it to undergo a series of conformational changes.Studies have shown that estrogen can inhibit RhoA/ROCK pathway expression in coronary,bladder,urethra,blood vessels and other smooth muscle.Our study would investigate the effect of RhoA/ROCK pathway expression on relaxation of rat colonic smooth muscle regulated by estrogen to provide a new target for the treatment of estrogen-related constipation and other gastrointestinal motility disorders.Objectives 1.To investigate the expression of RhoA/ROCK pathway in rat colonic smooth muscle regulated by 17?-estrodiol.2.To examine the effect of 17?-estradiol on the expression of RhoA/ROCK pathway in rat colonic smooth muscle cells(SMCs).Methods 1.Animal experiments(1)Female Sprague-Dawley(SD)rats were randomly divided into 4 groups,and all groups were under ovariectomy before.A 30-mm silicone tube filled with different solution was subcutaneously implanted in different groups of rats.Group C was filled with the solvent;Group E2 was E2 in corn oil(0.3 mg/m L)which can keep serum levels of E2 at physiological levels(56-92 pg/m L);Group EI was ICI 182780(estrogen receptor [ER] antagonist)plus E2 and group BSA-E2 was with BSA-E2.(2)After 14 days of intervention,the rats were killed and the colon muscle strips were taken.The contraction strength of rat muscle strips was measured.The distribution and expression of RhoA/ROCK pathway related molecules were detected.2.Cell experiments(1).Expression of ROCK1 and estrogen receptor in colonic SMCs: Double-immunohistochemical staining of rat colonic SMCs for ?-Actin,ROCK1 and estrogen receptors.(2).Effect of E2 on the expression of RhoA/ROCK in colonic SMCs:(1)Colonic SMCs were cultured with different concentrations of E2 for 24 h or with 50nmol/L E2 for different periods of time.The expression of RhoA,ROCK1 in colonic SMCs were measured by q RT-PCR and Western blot.(2)The downstream protein expression was detected by Western blot after 24 h stimulation with different concentrations of ROCK inhibitor Y27632 plus 50nmol/L E2.(3).Types of E2 receptors affecting RhoA/ROCK pathway:(1)Colonic SMCs were treated with DMSO,E2,ER? selective agonist propyl pyrazole triol(PPT),ER? selective agonist diaryl propiolnitrile(DPN),ICI 182780(estrogen receptor inhibitor)plus E2 or Albumin Bovine Serum conjugated 17?-estradiol(BSA-E2),and then the expression of RhoA and ROCK1 were measured by q RT-PCR and Western blot.(2)The ER? and ER? si RNAs were constructed and transfected into rat colonic SMCs.The expression of RhoA and ROCK1 were detected by q RT-PCR and Western blot after E2 stimulation.Results 1.Contraction of muscle strips in each group: The constraction of group E2 was significantly lower than that in other groups(P<0.05);There was no significant difference between group EI,group BSA-E2 and group C.2.Expression of RhoA and ROCK1 in muscle strips of each group: Compared with the other groups,the expression of RhoA and ROCK1 in colonic muscle strips in group E2 was significant decreased.There was no significant difference between group EI,group BSA-E2 and group C.3.Double immunofluorescence staining showed that ROCK1 and estrogen receptors are co-expressed with ?-Actin,respectively,in colonic SMCs.4.E2 treatment significantly decreased the expression of RhoA and ROCK1.The optimum concentration and time were 50nmol/L,24 h,respectively.The ROCK inhibitor Y27632 can inhibit downstream protein expression in a concentration-dependent manner.5.The expression of RhoA and ROCK1 was significantly inhibited by estrogen receptor agonist(PPT)(P<0.05),while it was not affected by estrogen receptor agonist(DPN)and BSA-E2.E2 can up-regulate the expression of RhoA and ROCK1 after interference by estrogen alpha receptor si RNA.Conclusions 1.Estrogen inhibits colonic smooth muscle contraction and expression of RhoA/ROCK pathway related molecules in colonic smooth muscle.2.E2 inhibits the expression of RhoA/ROCK pathway in rat colonic SMCs,which is mediated by estrogen receptor alpha.