| Perimenopausal syndrome (PPS) is a systematic disease occurred in perimenopausal women include imbalance in the spirit of the nemo-psychological, endocrine and metabolic, attributing to the change of the central and peripheral nerve neurotransmitter, vasomotor factor, cytokines free radicals. Liver-Qi stagnation plays an important role in perimenopausal syndrome, the priority of treatment is to soothe the liver and regulate the Qi and dispel the stagnation, such as Chaihu-Shugan-San(CSS), this study from the clinical and animal experiments explore the TCM pathological feature of perimenopausal syndrome and the relationship of Liver-Qi stagnation syndrome and strogen, estrogen receptors and monoamine neurotransmitters expression, to explore the relationship of gene expression in Ca2+/cAMP signaling pathway.ObjectiveTo explore the Liver-Qi stagnation syndrome’s TCM pathology and syndrome element mixed characteristics in PPS. To observe the relationship of Liver-Qi Stagnation score change and estrogen, neurotransmitters after treating with CSS in PSS Liver-Qi stagnation syndrome rats and to explore the biology basis of Liver-Qi stagnation in PPS.MethodsWe collected161patients of PPS to analyse the main locations and characteristic of the disease throught differentiation of syndrome elements method and syndrome differentiation to analyse main location and characteristic of the disease.100cases of PPS whose Liver-Qi Stagnation score exceed70are collected, after4weeks treatment with CSS, the relationship of Liver-Qi Stagnation score change and estrogen, neurotransmitters are observed. To observe the effective of CSS on PPS and Liver-Qi stagnation syndrome rat models, and explore the molecular mechanisms throught Ca2+/cAMP signaling pathway in hippocampal cell. In this experiments, twelve-week-old female rats (250-280g, n=12) are used as normal controls (Group A),52-week-old nature aging rat as PPS rat. A total of48nature aging rats (52-week-old,370-380g) are randomly assigned into four groups; PPS controls (Group B, n=12), PPS controls treate with CSS (Group C, n=12), PPS+Liver-Qi stagnation model treate with CSS (Group D, n=12), and PPS+Liver-Qi stagnation model (Group E, n=12). CSS is administered simultaneously when the Liver-Qi stagnation model being established. We selecte the sucrose consumption test and the open field test (OFT) for the Liver-Qi stagnation behavior test.We measure E2, neurotransmitters, CaMKII levels in serum and hippocampus by ELISA, Ca2+is measured by Fura-2/AM in hippocampus and estrogen receptors of hippocampal mRNA expression are measured by quantitative real-time PCR and Ca2+/cAMP signaling pathway by RT2Profiler PCR Arrays.ResultThe part of clinical Study1.The clinical symptoms distribution frequency of the top ten are hot flashes, sweating, irritability, depression or anxiety, menstrual disorders, chest distress, deep sigh, palpitations, globus hystericus, that is91.93%,91.93%,85.09%,81.99%,78.26%,69.56%,67.70%,55.28%,52.17%,39.75%.2.The locations of PPS are liver, kidney, spleen, heart, but mainly in the liver(91.93%) and kidney(52.17%), P<0.01.The score of liver locations is the highest.3. The nature syndrome elements include Qi stagnation and Yin deficiency, Yang deficiency, blood deficiency. Qi stagnation and Yin deficiency are in a high proportion, the ration is90.06%and71.43%。 The score of Qi stagnation is the highest.4. The mixed syndrome elements relationship of the locations mixed mainly are liver and kidney, spleen and kidney, liver and spleen, nature of mixed syndrome elements are Qi stagnation mixed Yin deficiency and Qi stagnation mixed blood deficiency.5.Before treatment, the cases of pathological Liver-Qi stagnation grade1is50, grade2is34, grade3is16, after treatment, the cases of pathological Liver-Qi stagnation grade0is70, grade1is22, grade2is3, grade3is5. The pathological classification changes significantly,P<0.01.6. Before and after treatment with CSS, the Kupperman score change significantly P <0.01.7. Before and after treatment with CSS, there is significant correlation relationship between Liver-Qi stagnation score and estrogen expression, r=-0.479P=0.001.8. Before and after treatment with CSS, there is significant correlation relationship between Liver-Qi stagnation score and neurotransmitters expression,(5-HT, R=-0.417,P=0.001;NE, R=-0.352P=0.001;DA, R=-0.609P=0.000)Part II The part of Basical Study1. The open field test and the sucrose consumption test, the dynamic changes of BW. The BW of group E decrease sharply, while that of group D decreased slowly. We found the sucrose intake in group E are significantly lower than that in group B, p<0.01), whereas the CSS treatment ameliorate the sucrose intake (group D vs. group E, p<0.01).2. CSS administration effects on hippocampal ERa, ERβ, or GPR30mRNA expression in aging rats or Liver-Qi stagnation group rats.No significant difference is observed in ERβ mRNA expression between the four groups, thereby indicating that CSS administration does not affect the ERβ mRNA expression in groups B and C or groups D and E. Similarly, GPR30mRNA expression does not differ significantly between treatment groups. The ERa/ERβ ratio in group E is markedly decrease compared with the other groups, p<0.01. CSS administration increase ERa/ERβ ratio.E2levels in A group rats were significantly higher than those in the group B. No significant difference is observed between the four aging rat groups, indicating that CSS does not affect E2levels in aging rats. Hippocampal5-HT, NE, DA levels in A group are significantly higher than those aging group, after CSS treatment NE, DA levels increase. The change of serum NE and DA are the same as of hippocampus except5-HT.3. The E group hippocampal Ca2+expression increase sharply than D group, P<0.01. The change of CaMKII expression is the same.4. PCR Arrays indicate there are6functional gene namely S100g, Th, NOS2, IL-2, Calml and SSt expression change between E group and B group (P<0.05or P<0.01), after CSS administration only the SSt functional gene change P<0.05.Conclusion1. The main clinical symptoms of PSS is Liver-Qi stagnation symptoms.The main locations of PPS are liver. The characteristic of syndrome elements in PPS is multiple organ involvement, mixed excessiveness and deficiency. Liver-Qi stagnation is the main pathology. The main clinical symptoms of the PSS are hot flashes, sweating, irritability, depression or anxiety, menstrual disorders, chest distress, deep sigh, palpitations, globus hystericus.2. There are significant differences in Liver-Qi Stagnation score and Kupperman score before and after treatment. There is significant difference between Liver-Qi Sagnation score and estrogen before and after treatment. There are significant negative correlation between the pathology grade and estrogen. There are significant differences between Liver-Qi stagnation score and neurotransmitters5-HT, NE, DA before and after treatment. There are significant negative correlation between the pathology grade and neurotransmitters5-HT, NE, DA.3. The open field test and the sucrose consumption test, the dynamic changes of BW and E2content in serum and hippocampus verify the PPS+Liver-Qi Stagnation syndrome rats model is good.4. It indicates that there is close relation between ERa/ERβ ratio and Liver-Qi Stagnation syndrome in PPS rats. There is close relation between neurotransmitters and Liver-Qi stagnation syndrome in PPS.5. There is close relation between Liver-Qi stagnation syndrome and Ca2+/CaMKII signal pathway in PPS. There is close relation between genes relating to neurotransmitter in Ca2+/cAMP signaling pathway and Liver-Qi stagnation syndrome, maybe it was the molecular mechanisms of Liver-Qi stagnation syndrome of PPS. |
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