ABSTRACT
OBJECTIVE To investigate the mechanism of Mayuan tongbian zhitong decoction on improving slow-transmission constipation（STC）in rats by regulating AMP activated protein kinase （AMPK）/endothelial nitric oxide synthase （eNOS）signaling pathway. METHODS The rats were randomly divided into blank group ，model group ，Mayuan tongbian zhitong decoction low-dose，medium-dose and high-dose groups （6，12，18 mg/kg），with 10 rats in each group. Except for blank group ，other groups were given Compound diphenoxylate suspension to induce STC model. After modeling ，blank group and model group were given normal saline intragastrically ，and Mayuan tongbian zhitong decoction groups were given relevant medicine intragastrically ， once a day ，for consecutive 2 weeks. The number of feces and water content of feces in each group were observed before and after treatment；the carbon powder propulsion rate of rats in each group was calculated ；the pathological structure of colon in each group was observed ；the levels of nitric oxide （NO）and nitric oxide synthase （NOS）in colon tissues of rats in each group were detected；the expressions of AMPK ，eNOS，mammalian target of rapamycin （mTOR），tuberous sclerosis complex 1（Tsc-1）， Tsc-2 and eukaryotic promoter 4E binding protein （4ebp）were also detected. The active ingredients of Cannabis sativa ，Citrus aurantium and Rehmannia glutinosa were screened from Mayuan tongbian zhitong decoction. The active ingredients with high Degree value were docked with AMPK and eNOS ，to verify the interaction. RESULTS Compared with before treatment ，the number and water content of feces were increased significantly in Mayuan tongbian zhitong decoction groups （P＜0.05）. Compared with blank group ，carbon powder propulsion rate of model group was decreased significantly （P＜0.05）； colonic structure was disordered ，and a large number of inflammatory cells were seen in submucosa ；the levels of NO and NOS in colon tissue as well as the protein expressions of AMPK ，eNOS，mTOR，Tsc-1，Tsc-2 and 4ebp were increased significantly （P＜0.05）. Compared with model group ，above indexes of Mayuan tongbian zhitong decoction groups （except for NOS in low-dose group ）were reserved significantly （P＜0.05）. In the molecular docking experiment ，the active components with the highest Degree values in C. sativa ，C. aurantium and R. glutinosa were（Z）-3-（4-hydroxy-3-methoxy-phenyl）-N-[2-（4-hydroxyphenyl）ethyl] acrylamide，nobiletin and stigmasterol. The binding energies of AMPK with these three components were －5.15，－4.61 and －4.83 kJ/mol，the binding energies of eNOS with these three components were －6.11，－5.40 and －5.91 kJ/mol. The conformations of these three compounds with AMPK and eNOS were stable and their binding activities were high. CONCLUSIONS Mayuan tongbian zhitong decoction can improve the constipation symptoms and intestinal function in STC model rats ，and its specific mechanism may be related to the inhibition of AMPK/eNOS signaling pathway.
ABSTRACT
Obej ctive To investigate the role of metastasis associated protein 1(MTA1)in estrogen reg-ulated expression of matrix metalloproteinase -9(MMP-9)and tissue inhibitor of metalprotease -1(TIMP-1) in estrogen receptor( ER ) positive breast cancer cells .Methods MTA1 knockdown cell model was generated based on MCF-7breast cancer cell line by transfected with MTA 1-shRNA.The mRNA and protein level of MMP-9 and TIMP-1 in wild type MCF-7(MCF-7WT)and MCF-7MTA1-shRNA before and after 17β-estradiol ( E2) treatment were examined by Real -time PCR and Western blot respectively .Results The MTA1-shRNA showed maximally 84.9%suppression of MTA1 expression in MCF-7,suggesting a satisfied MTA 1 knockdown cell model was established for subsequent experiments .After treated with E2 for 48 h,MCF-7WT showed an incre-ment of 46%(P<0.05)and 37%(P<0.05)of the mRNA and protein level of MMP -9 and a decrement of 32.3%( P<0.05)and 18.2%(P<0.05)of TIMP-1;MCF-7MTA1-shRNA showed a decrement of 32.3%(P<0.05)and 18.2%(P<0.05)of mRNA and protein expression of MMP -9 respectively but no significant differ-ence in TIMP-1 comparing with MCF-7WT before treated with Estradiol.After E2 treatment,MCF-7MTA1-shRNA didn′t show significant change of MMP -9 except decrements of 32.3%(P<0.05)and 18.2%(P<0.05)in the mRNA and protein levels of TIMP -1.Conclusion MTA1 may be involved in the pathway by which estrogen regulated the expression of MMP -9 but not TIMP-1 in ER positive breast cancer cells .