| Objective:To study the rat model of premenstrual syndrome with liver qi stagnation at different period of receptive phases and non-receptive phases,and explore the Shuyu capsule effects on various characteristic changes of GABA-T?GAD?GAT-1 in different brain regions,and then explore the mechanism of Shuyu capsule in the treatment of remenstrual syndrome with liver qi stagnation syndrome.Methods: 1.Prepare female rats with regular estrous cycle by vaginal smears,according to body weight,open field distance of the rats,the rats were randomly divided into normal control group,stress group,stress + shuyu capsule group(SY group),stress + fluoxetine group(FXT group).The rat model of premenstrual syndrome with liver qi stagnation syndrome was prepared by the improved optimized chronic restraint stress method in two estrous cycles.After the stress,the acquisition of behavior of the rats: body mass index,total open field distance and forced swimming suspension immobility time,then rats in each group is determined according to the period of vaginal smear,peripheral blood and brain regions : hippocampus,frontal,parietal,hypothalamus,stored in the-70 C to be detected.2.ELISA kit was used to detect the content of Glu(glutamic acid,glutamate),GABA(gamma aminobutyric acid,gamma-aminobutyric acid)in peripheral blood of rats in the period of receptive phases and non non-receptive phases.3.Berthelot method was used to detect the activity of glutamate decarboxylase(GAD)and gamma aminobutyric acid transaminase(GABA-T)in different brain regions(hippocampus,frontal lobe,parietal area and hypothalamus)of rats in the period of receptive phases and non non-receptive phases.4.Real time fluorescent quantitative PCR and Western blot was used to detect themRNA and proteinin expression of GABA-T,GAD,GAT-1 in different brain regions(hippocampus,frontal lobe,parietal area and hypothalamus)of rats in the period of receptive phases and non non-receptive phases.Results: 1.The evaluation of rats model: body weight gain: after the stress,compared with normal control group,body weight gain of rats in stress group were significantly decreased(P < 0.05);compared with the stress group,after given drug intervention,FXT and SY group were significantly increased(P < 0.05).The open field test: compared with normal control group,stress group rats in the open field distance was significantly decreased(P < 0.05);After given drug intervention,compared with the stress group,open field distance of FXT group and SY group were significantly increased(P < 0.05).The suspension time: compared with normal control group,the suspended time in stress group was significantly increased(P <0.05),compared with the stress group,after given drug intervention,the suspended time in FXT group and SY group suspension was significantly decreased(P < 0.05).2.The concentration of Glu and GABA in rats with different estrous cycle: At period of non-receptive phases,compared with the normal control group,the concentration of Glu in stress group was significantly increased(P < 0.05),compared with the stress control group,the concentration of Glu in FXT group and SY group was significantly decreased(P < 0.05);Compared with the normal control group,the concentration of GABA in stress group was significantly decreased(P < 0.05),compared with the stress control group,the concentration of GABA in FXT group and SY group was significantly increased(P < 0.05);At period of receptive phases,there was no significant difference in Glu and GABA levels between the two groups(P>0.05).3.The GABA-T,GAD enzyme activity of different estrous cycle:through detection,at period of receptive phases and non-receptive phases of different brain regions(hippocampus,frontal lobe,parietal area and hypothalamus),there were no significant differences in different groups(P>0.05).4.The mRNA and proteinin expression of GABA-T,GAD,GAT-1 in different estrous cycle of rats:(1)The mRNA expression:At period of receptive phases,the expression of GABA-T,GAD,GAT-1 in different brain regions(hippocampus,frontal lobe,parietal area and hypothalamus)showed no significant difference(P>0.05).At period of non-receptive phases,compared with the normal controlgroup,stress group of the m RNA expression of GABA-T,GAT-1 in different brain regions(hippocampus,frontal lob,hypothalamus)was significantly decreased(P<0.05),tress group of the expression of GABA-T,GAT-1 in the brain of parietal area was significantly increased(P<0.05),the expression of GAD65,GAD67 in different brain regions(hippocampus,frontal lob,parietal area,hypothalamus)was significantly increased(P<0.05);Compared with the stress group,stress + FXT group and SY capsule group of the mRNA expression of GABA-T,GAT-1 in different brain regions(hippocampus,frontal lobe,hypothalamus)was significantly increased(P<0.05),parietal area was significantly decreased(P<0.05),the mRNA expression of GAD65,GAD67 in different brain regions(hippocampus,frontal lob,parietal area,hypothalamus)significantly decreased(P<0.05).(2)The protein expression: At period of receptive phases,the protein expression of GABA-T,GAD,GAT-1 in different brain regions(hippocampus,frontal lobe,parietal area,hypothalamus)showed no significant difference(P>0.05).At period of non-receptive phases,compared with the normal control group,stress group of the protein expression of GABA-T,GAT-1 in different brain regions(hippocampus,frontal lob,hypothalamus)was significantly decreased(P<0.05),stress group of the expression of GABA-T,GAT-1 in the brain of parietal area was significantly increased(P<0.05),the protein expression of GAD65,GAD67 in different brain regions(hippocampus,frontal lob,parietal area,hypothalamus)was significantly increased(P<0.05);Compared with the stress group,FXT group and SY capsule group of the protein expression of GABA-T,GAT-1 in different brain regions(hippocampus,frontal lobe,hypothalamus)was significantly increased(P<0.05),parietal area was significantly decreased(P<0.05),the protein expression of GAD65 in different brain regions(hippocampus,frontal lob,parietal area,hypothalamus)significantly decreased(P<0.05),the protein expression of GAD67 in different brain regions(hippocampus,frontal lob,hypothalamus)significantly decreased(P<0.05).Conclusion: By the improved optimized chronic restraint stress method can successfully establish the rat model of premenstrual syndrome with liver qi stagnation syndrome,the occurrence of the disease is related to the abnormal m RNA and protein expression of GABA-T,GAD,GAT-1 in different brain regions,which can cause the abnormal level of Glu and GABA,leading to the imbalance of Glu/GABA,and cause the disease;Shuyu capsule can correct the abnormalexpression of m RNA and protein,restore the balance of Glu/GABA,and to provide an new target for premenstrual syndrome with liver qi stagnation syndrome. |
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