疏肝解郁法对肝郁证模型大鼠的生物学基础研究
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摘要
目的：研究柴胡疏肝散对肝郁证模型大鼠的行为学影响,并从内分泌、免疫、细胞信号转导通路等方面探讨柴胡疏肝散治疗肝郁证的作用机制,进而揭示疏肝解郁法治疗肝气郁结证的机制,为疏肝解郁法治疗肝郁证提供理论依据及实验基础。
方法：
1肝郁大鼠模型的建立：将大鼠选用开野实验(Openfield test)将评分相近的大鼠随机分为正常组和模型组,每组40只。运用模具束缚结合孤养的方法制备肝气郁结证侯动物模型,共4周,以周为单位观察不同时间点大鼠外观行为的变化；放免法测定血清CORT、GAS、IL-2,血浆ACTH、MTL;称量大鼠体重及免疫器官脾、胸腺的重量。
2柴胡疏肝散对肝郁证模型大鼠作用的比较：选用开野实验(Openfield test)将评分相近的大鼠随机分为正常组,模型组,柴胡疏肝散组(疏肝解郁法)和四君子汤组(益气健脾法),每组10只。模型组动物按前述束缚法结合孤养法制模二周,正常对照组不做处理,自然饲养。从造模之日起模型组和正常组分别给予等量生理盐水灌胃每日一次；柴胡疏肝散组和四君子汤组动物分别按1ml／100g给予相应药液灌胃每日一次。服药时间为2周。于末次给药2h后观察各组大鼠的行为学改变,包括开野实验与强迫游泳实验。各组大鼠于造模两周后次日取材,指标测定同上。
3柴胡疏肝散对肝郁证模型大鼠ERK-CREB信号转导通路免疫组织化学研究：造模与给药同上,各组大鼠于造模两周后次日取材,采用免疫组化方法观察各组大鼠脑组织海马CA1、CA3、DG和BLA区ERK1/2、P-ERK. CREB、P-CREB的免疫阳性神经元的细胞数和面积。
结果：
1与正常组比较,模型组在造模期间,大鼠体重降低,第一至第四周均有显著差异(P<0.001)；血清GAS下降,第二周和第三周有显著差异(P<0.05)；血浆MTL下降,第二周到第四周有显著差异(P<0.05)；血浆ACTH、血清CORT升高,第二至第四周均出现显著差异(P<0.05)；血清GAS、血浆MTL含量升高,第三周有显著差异(P<0.05)；脾重量和胸腺重量四周均降低并有显著性差异(P<0.01或P<0.001)；血清免疫因子IL-2下降,第一至第四周有显著差异(P<0.01或P<0.05)。
2与正常组比较,模型组在造模期间体重增加缓慢,有显著性差异(P<0.001)；开野实验中的水平运动、垂直运动得分明显下降(P<0.01),中央格停留时间与粪便粒数有上升的趋势(P>0.05)；大鼠强迫游泳的不动时间明显延长(P<0.001)；血清CORT和血浆ACTH含量升高(P<0.001)；血浆MTL和血清GAS含量下降(P<0.05)；脾重量、胸腺重量明显下降(P<0.01,P<0.001)；血清IL-2含量明显下降(P<0.01)。
与模型组比较,柴胡疏肝散组体重增长明显,有显著性差异(P<0.001),四君子汤组体重增长差异性显著(P<0.001)；柴胡疏肝散组大鼠水平得分及垂直得分均明显增高(P<0.05),中央格停留时间与粪便粒数有减少的趋势(P>0.05),四君子汤组水平得分及垂直得分均增高,中央格停留时间与粪便粒数有减少的趋势,但无差异(P>0.05)；柴胡疏肝散组大鼠强迫游泳的不动时间明显缩短(P<0.001),而四君子汤组大鼠强迫游泳的不动时间未见明显缩短,无显著性差异(P>0.05)；柴胡疏肝散组大鼠血清CORT和血浆ACTH含量下降(P<0.01,P<0.001),而四君子汤组大鼠血清CORT和血浆ACTH含量稍有下降,无显著性差异(P>0.05)；柴胡疏肝散组大鼠血浆MTL和血清GAS含量增高(P<0.05),而四君子汤组大鼠血浆MTL和血清GAS含量有增高趋势,无显著性差异(P>0.05)；二方组大鼠脾重量、胸腺重量均增高(P<0.05,P<0.01)；二方组大鼠血清IL-2含量均增高(P<0.05)。
与正常组比较,柴胡疏肝散组体重增长无明显差异(P>0.05),四君子汤组体重增长缓慢,有显著性差异(P<0.001)；二方组大鼠水平得分及垂直得分均增高,中央格停留时间与粪便粒数有减少的趋势,但无显著差异(P>0.05)；柴胡疏肝散组大鼠强迫游泳的不动时间明显缩短无明显差异(P>0.05),四君子汤组大鼠强迫游泳的不动时间明显延长,有显著性差异(P<0.001)；二方组大鼠血清CORT和血浆ACTH含量下降无明显差异(P>0.05)；二方组大鼠血浆MTL和血清GAS含量增高无明显差异(P>0.05)；二方组大鼠脾重量、胸腺重量增高无明显差异(P>0.05)；二方组大鼠血清IL-2含量均增高无明显差异(P<0.05)。
3与正常组比较,模型组ERK1/2、P-ERK在海马CA3、DG区和BLA区阳性细胞数明显减少,ERK1在海马CA3区、ERK2在海马CA3区与BLA区、P-ERK在海马DG区和BLA区阳性面积明显减少；CREB、P-CREB在海马各区与BLA区阳性细胞数、阳性面积明显减少。
与模型组比较,柴胡疏肝散组大鼠ERK1/2、P-ERK在海马CA3、DG区和BLA区阳性细胞数明显增多,ERK1在海马CA3区、ERK2在海马CA3区与BLA区、P-ERK在海马DG区和BLA区阳性面积明显增多,而四君子汤组大鼠ERK1/2、P-ERK在海马各区和BLA区阳性细胞数、阳性面积有增多趋势,但无显著差异；柴胡疏肝散组大鼠CREB、P-CREB在海马各区与BLA区阳性细胞数、阳性面积显著增多,而四君子汤组大鼠海马各区和BLA阳性细胞数、阳性面积有增多趋势,但无显著差异。
与正常组比较,ERK1/2、P-ERK、CREB、P-CREB在柴胡疏肝散组与四君子汤组海马各区和BLA区阳性细胞数、阳性面积均无统计学差异。
结论：
1本实验侧重从内分泌、免疫系统和胃肠激素角度观察了复制肝郁证模型不同时间点的相关指标的动态变化,认为用束缚法结合孤养法造模两周即可复制出肝郁证大鼠模型。
2疏肝解郁法的代表方剂柴胡疏肝散可通过增加肝郁证大鼠的水平与垂直运动得分,延长中央格停留时间,增多粪便粒数,缩短强迫游泳的不动时间,对肝郁大鼠的行为起到显著改善作用。
3疏肝解郁法的代表方剂柴胡疏肝散具有抗肝郁作用,其机制可能与其调节肝郁大鼠血液ACTH和CORT的水平,对HPA轴有抑制作用有关,柴胡疏肝散还可通过升高肝郁大鼠血浆MTL和血清GAS含量来调节胃肠功能,通过升高肝郁大鼠血清IL-2含量、脾／胸腺重量改善机体免疫功能,从而有效发挥抗肝郁作用。
4疏肝解郁法的代表方剂柴胡疏肝散具有抗肝郁作用,其机制可能与通过上调肝郁大鼠海马CA3、DG区和BLA区ERK1/2、P-ERK信号通路的活动而起到保护受损神经元,改善大脑功能,缓解肝郁症状的作用。柴胡疏肝散还可通过上调肝郁大鼠海马各区与BLA区CREB、P-CREB的表达,促进应激损伤神经元的恢复,改善大鼠的肝郁症状,从而有效发挥抗肝郁作用。
5疏肝解郁法治疗肝郁证的机制可能是通过调节血液ACTH和CORT的水平,对HPA轴有抑制作用有关,通过升高血浆MTL和血清GAS含量来调节胃肠功能,通过升高血清IL-2含量、脾／胸腺重量改善机体免疫功能；通过上调海马CA3、DG区和BLA区ERK1/2、P-ERK信号通路的活动而起到保护受损神经元,通过上调海马各区与BLA区CREB、P-CREB的表达,促进应激损伤神经元的恢复。ERK-CREB这一信号通路是疏肝解郁法治疗肝郁证发挥作用的关键靶点。
Objective:To investigate the effect of CHSGS on the example rats which suffer Liver-Qi Stagnation Syndrome, plus with the study of CHSGS effect on Liver-Qi Stagnation Syndrome through the way of endocrine, immunization and signal transduction pathways, it reveals the way of relieving depression of the liver-qi and sets a basic study on relieving depression of the liver-qi.
Method:
1 The set up of liver-qi depression rats:we divide those rats into regular group and model group by adopting Open field test, forty each. It takes four weeks for them to infect liver-qi depression by keeping them alone and tools constraints-breeding. We observe those rats appearance weekly; detecting blood serum CORT, GAS, IL-2, blood plasma ACTH,MTL and weighing rats weight, spleen and thymus gland.
2 Compared with the Chaihushugan San group:the rats are divided into regular groups, model group, CHSGS group and SJZT Group by adopting Openfield test, ten each. The model group will be bred two weeks by keeping them alone and tools constraints. The regular group and model group will be physiological saline lavaged once a day. CHSGS group and SJZT group would be lavaged lml/100g once a day. Two weeks later, we observed each group on behavioral biology including Open field test and Forced Swimming test after the two hours later after the last lavage. Then we observe the actions of rats, use RAImmune to detect blood serum CORT、GAS. IL-2, blood plasm MTL; weigh body mass of rat and weight of spleen and thymic.
3 The Chemistry study of CHSGS group and liver-qi depression group ERK-CREB cell Signaling Pathway:we used the same way which as mentioned above to breed them, then killed the rats the day after two weeks. those model groups would be obtained two weeks later and observed brain organization Hippocampus japonicus CA1、CA3、DG and BLA section CREB. P-CREB. ERK1/2. P-ERK cell number and immune positive neuron.
Results:
1 Compared with the regular group, during making models, the rats are losing weights. There were big differences from week one to week four (P<0.001); blood serum decrease as well, and also the big differences between week two and week three (P<0.05) as well as week two to week four (P<0.05). Plasma ACTH and blood serum CORT increased between week two till week four. Blood serum and plasma increased at week three. Spleen and thymus gland decrease at week four (P<0.01,P<0.001) and immuno-cell factor IL-2 decrease at week four.
2 Compared with regular group, during making model, the model group increasing weight slowly and big differences (p<0.001); horizontal movement and vertical movement during of Open field test decrease (p<0.01), blood serum CORT and plasma ACTH increases (p＜0.001); plasma MTL and blood serum GAS decreases (P＜0.05); spleen weight and thymus gland weights decrease (p<0.01, p<0.001); blood serum IL-2 decrease a lot (p<0.01)
Compared with model group, CHSGS group increases a lot weight (p<0.001), so does the SJZT group (p＜0.001); horizontal score and vertical score of CHSGS group increases and the stay time in the central form and rats'shit decreased. SJZT group encountered the similar situation but make no differences (p>0.05); the immobility time of forced swimming of CHSGS group shortened and The SJZT group didn't and made no obvious differences (p>0.05); the amount of blood serum CORT and plasm ACTH from CHSGS group decreased (p<0.01, p＜0.001), the amount of blood serum CORT and plasm ACTH from The SJZT decreased a bit and made no obviously differences (p>0.05); the amount of blood serum GAS and plasm MTL from CHSGS group increased (p>0.05), however, the amount of blood serum GAS and plasm MTL from The SJZT group tend to increase as well, and made no obviously differences (p>0.05); the weight of spleen and thymus from both group increased (p<0.05, p<0.01);and blood serum IL-2 from group increased as well (p<0.05).
Compared with regular group, body weight from CHSGS group increased but with no differences (p>0.05), body weight from SJZT group increased slowly and with a lot different (p<0.001>); horizontal scores and vertical scores from both group increased, stay time in central form and shit amount decreased, but made no obviously differences (p>0.05); the immobility time of forced swimming from CHSGS group decreased obviously (p>0.05), made no obviously differences. The immobility time of forced swimming from The SJZT group shortened a lot and made great differences (p>0.001); the amount of blood serum CORT and plasma ACTH from both group decreased and made no obvious differences (p>0.05); the amount of blood serum GAS and plasma MTL from both group increased and made no obvious differences (p>0.05); the spleen weight and thymus gland weight from both group increased and made no obvious differences (p>0.05); the amount of blood serum IL-2 increased and made no obvious differences (P<0.05).
3 Compared with regular group, CA3, DG section from Hippocampus japonicus and BLA ERK1 amount of positive cell, the positive area from CA3 section ERK1 of Hippocampus japonicus decreased a lot. CA3, DG section from Hippocampus japonicus and BLA ERK2 amount of positive cell, the positive area from CA3 section ERK2 of Hippocampus japonicus decreased a lot; CA3, DG section and BLA P-ERK positive cell amount from Hippocampus japonicus, DG section and BLA positive area decreased a lot.; each section of Hippocampus japonicus from model group and BLA CREB, P-CREB amount of positive cell, the positive area decreased a lot.
Compared with model group, the Hippocampus japonicus CA3, DG section and BLA ERK1/2 amount of positive cell from CHSGS, and Hippocampus japonicus CA3 section ERK1/CA and BLA ERK2 increased obviously, and the same thing happen to the SJZT Group but made no obvious differences. Hippocampus japonicus CA3, DG section and amount number of BLAP-ERK positive cell, Hippocampus japonicus DG section and BLA positive area increased, those figures from The SJZT tend to increase as well but with no obvious differences;the amount of BLA CREB, P-CREB positive cell, positive area from each section of Hippocampus japonicus increased obviously, the same thing happened to The SJZT group but didn't make a lot differences.
Compared with regular group, the above figures from CHSGS group and SJZT group has no differences.
Conclusion:
1 This experiments focus on internal secretion, immunization, stomach and Intestines to observe the way to produce the different circumstances of stagnation of liver-QI on different period, which indicates that it only takes two weeks that can produce stagnation of liver-QI rats'model.
2 CHSGS can make scores by increasing horizontal movement and vertical movement of stagnation of liver-QI rats. It can improve the stagnation of liver-QI rats' behavior by extending stay in the central form, increasing the shit amount, shortening the forced swimming time.
3 CHSGS has the power of anti stagnation of liver-QI; it is probably related to improve the rats'blood ACTH and CORT, which restrains HPA axle. CHSGS can also adjust stomach and intestines by improving plasm MTL and GAS. It mainly improves immunization system by increasing the amount of stagnation of liver-QI rats'blood serum IL-2, spleen weight and thymus gland weight, from which improved the power of anti stagnation of liver-QI.
4 CHSGS has the power of anti stagnation of liver-QI; CHSGS can protect the damaged nerve cell; improve the brain functions, relieving stagnation of liver-QI rats' symptom by improving Hippocampus japonicus CA3, DG section and BLA ERK1/2, P-ERK signal pathway activities;it is probably related to the improvement each section of rats'Hippocampus japonicus and BLA section CREB, P-CREB. It boosts the recovery of those stressed damaged nerve cell, improves the stagnation of liver-QI rats'symptom, moreover, and improves the power of anti stagnation of liver-QI.
5 The way of relieving depression of the liver-qi can relieve depression of the liver-qi by improving the blood ACTH and CORT, which restrains HPA axle,improve the plasm MTL and GAS, improving immunization system by increasing the amount of stagnation of blood serum IL-2, spleen weight and thymus gland weight, improving Hippocampus japonicus CA3, DG section and BLA ERK1/2, P-ERK signal pathway activities, improving each section of Hippocampus japonicus and BLA section CREB, P-CREB.
方法：
1肝郁大鼠模型的建立：将大鼠选用开野实验(Openfield test)将评分相近的大鼠随机分为正常组和模型组,每组40只。运用模具束缚结合孤养的方法制备肝气郁结证侯动物模型,共4周,以周为单位观察不同时间点大鼠外观行为的变化；放免法测定血清CORT、GAS、IL-2,血浆ACTH、MTL;称量大鼠体重及免疫器官脾、胸腺的重量。
2柴胡疏肝散对肝郁证模型大鼠作用的比较：选用开野实验(Openfield test)将评分相近的大鼠随机分为正常组,模型组,柴胡疏肝散组(疏肝解郁法)和四君子汤组(益气健脾法),每组10只。模型组动物按前述束缚法结合孤养法制模二周,正常对照组不做处理,自然饲养。从造模之日起模型组和正常组分别给予等量生理盐水灌胃每日一次；柴胡疏肝散组和四君子汤组动物分别按1ml／100g给予相应药液灌胃每日一次。服药时间为2周。于末次给药2h后观察各组大鼠的行为学改变,包括开野实验与强迫游泳实验。各组大鼠于造模两周后次日取材,指标测定同上。
3柴胡疏肝散对肝郁证模型大鼠ERK-CREB信号转导通路免疫组织化学研究：造模与给药同上,各组大鼠于造模两周后次日取材,采用免疫组化方法观察各组大鼠脑组织海马CA1、CA3、DG和BLA区ERK1/2、P-ERK. CREB、P-CREB的免疫阳性神经元的细胞数和面积。
结果：
1与正常组比较,模型组在造模期间,大鼠体重降低,第一至第四周均有显著差异(P<0.001)；血清GAS下降,第二周和第三周有显著差异(P<0.05)；血浆MTL下降,第二周到第四周有显著差异(P<0.05)；血浆ACTH、血清CORT升高,第二至第四周均出现显著差异(P<0.05)；血清GAS、血浆MTL含量升高,第三周有显著差异(P<0.05)；脾重量和胸腺重量四周均降低并有显著性差异(P<0.01或P<0.001)；血清免疫因子IL-2下降,第一至第四周有显著差异(P<0.01或P<0.05)。
2与正常组比较,模型组在造模期间体重增加缓慢,有显著性差异(P<0.001)；开野实验中的水平运动、垂直运动得分明显下降(P<0.01),中央格停留时间与粪便粒数有上升的趋势(P>0.05)；大鼠强迫游泳的不动时间明显延长(P<0.001)；血清CORT和血浆ACTH含量升高(P<0.001)；血浆MTL和血清GAS含量下降(P<0.05)；脾重量、胸腺重量明显下降(P<0.01,P<0.001)；血清IL-2含量明显下降(P<0.01)。
与模型组比较,柴胡疏肝散组体重增长明显,有显著性差异(P<0.001),四君子汤组体重增长差异性显著(P<0.001)；柴胡疏肝散组大鼠水平得分及垂直得分均明显增高(P<0.05),中央格停留时间与粪便粒数有减少的趋势(P>0.05),四君子汤组水平得分及垂直得分均增高,中央格停留时间与粪便粒数有减少的趋势,但无差异(P>0.05)；柴胡疏肝散组大鼠强迫游泳的不动时间明显缩短(P<0.001),而四君子汤组大鼠强迫游泳的不动时间未见明显缩短,无显著性差异(P>0.05)；柴胡疏肝散组大鼠血清CORT和血浆ACTH含量下降(P<0.01,P<0.001),而四君子汤组大鼠血清CORT和血浆ACTH含量稍有下降,无显著性差异(P>0.05)；柴胡疏肝散组大鼠血浆MTL和血清GAS含量增高(P<0.05),而四君子汤组大鼠血浆MTL和血清GAS含量有增高趋势,无显著性差异(P>0.05)；二方组大鼠脾重量、胸腺重量均增高(P<0.05,P<0.01)；二方组大鼠血清IL-2含量均增高(P<0.05)。
与正常组比较,柴胡疏肝散组体重增长无明显差异(P>0.05),四君子汤组体重增长缓慢,有显著性差异(P<0.001)；二方组大鼠水平得分及垂直得分均增高,中央格停留时间与粪便粒数有减少的趋势,但无显著差异(P>0.05)；柴胡疏肝散组大鼠强迫游泳的不动时间明显缩短无明显差异(P>0.05),四君子汤组大鼠强迫游泳的不动时间明显延长,有显著性差异(P<0.001)；二方组大鼠血清CORT和血浆ACTH含量下降无明显差异(P>0.05)；二方组大鼠血浆MTL和血清GAS含量增高无明显差异(P>0.05)；二方组大鼠脾重量、胸腺重量增高无明显差异(P>0.05)；二方组大鼠血清IL-2含量均增高无明显差异(P<0.05)。
3与正常组比较,模型组ERK1/2、P-ERK在海马CA3、DG区和BLA区阳性细胞数明显减少,ERK1在海马CA3区、ERK2在海马CA3区与BLA区、P-ERK在海马DG区和BLA区阳性面积明显减少；CREB、P-CREB在海马各区与BLA区阳性细胞数、阳性面积明显减少。
与模型组比较,柴胡疏肝散组大鼠ERK1/2、P-ERK在海马CA3、DG区和BLA区阳性细胞数明显增多,ERK1在海马CA3区、ERK2在海马CA3区与BLA区、P-ERK在海马DG区和BLA区阳性面积明显增多,而四君子汤组大鼠ERK1/2、P-ERK在海马各区和BLA区阳性细胞数、阳性面积有增多趋势,但无显著差异；柴胡疏肝散组大鼠CREB、P-CREB在海马各区与BLA区阳性细胞数、阳性面积显著增多,而四君子汤组大鼠海马各区和BLA阳性细胞数、阳性面积有增多趋势,但无显著差异。
与正常组比较,ERK1/2、P-ERK、CREB、P-CREB在柴胡疏肝散组与四君子汤组海马各区和BLA区阳性细胞数、阳性面积均无统计学差异。
结论：
1本实验侧重从内分泌、免疫系统和胃肠激素角度观察了复制肝郁证模型不同时间点的相关指标的动态变化,认为用束缚法结合孤养法造模两周即可复制出肝郁证大鼠模型。
2疏肝解郁法的代表方剂柴胡疏肝散可通过增加肝郁证大鼠的水平与垂直运动得分,延长中央格停留时间,增多粪便粒数,缩短强迫游泳的不动时间,对肝郁大鼠的行为起到显著改善作用。
3疏肝解郁法的代表方剂柴胡疏肝散具有抗肝郁作用,其机制可能与其调节肝郁大鼠血液ACTH和CORT的水平,对HPA轴有抑制作用有关,柴胡疏肝散还可通过升高肝郁大鼠血浆MTL和血清GAS含量来调节胃肠功能,通过升高肝郁大鼠血清IL-2含量、脾／胸腺重量改善机体免疫功能,从而有效发挥抗肝郁作用。
4疏肝解郁法的代表方剂柴胡疏肝散具有抗肝郁作用,其机制可能与通过上调肝郁大鼠海马CA3、DG区和BLA区ERK1/2、P-ERK信号通路的活动而起到保护受损神经元,改善大脑功能,缓解肝郁症状的作用。柴胡疏肝散还可通过上调肝郁大鼠海马各区与BLA区CREB、P-CREB的表达,促进应激损伤神经元的恢复,改善大鼠的肝郁症状,从而有效发挥抗肝郁作用。
5疏肝解郁法治疗肝郁证的机制可能是通过调节血液ACTH和CORT的水平,对HPA轴有抑制作用有关,通过升高血浆MTL和血清GAS含量来调节胃肠功能,通过升高血清IL-2含量、脾／胸腺重量改善机体免疫功能；通过上调海马CA3、DG区和BLA区ERK1/2、P-ERK信号通路的活动而起到保护受损神经元,通过上调海马各区与BLA区CREB、P-CREB的表达,促进应激损伤神经元的恢复。ERK-CREB这一信号通路是疏肝解郁法治疗肝郁证发挥作用的关键靶点。
Objective:To investigate the effect of CHSGS on the example rats which suffer Liver-Qi Stagnation Syndrome, plus with the study of CHSGS effect on Liver-Qi Stagnation Syndrome through the way of endocrine, immunization and signal transduction pathways, it reveals the way of relieving depression of the liver-qi and sets a basic study on relieving depression of the liver-qi.
Method:
1 The set up of liver-qi depression rats:we divide those rats into regular group and model group by adopting Open field test, forty each. It takes four weeks for them to infect liver-qi depression by keeping them alone and tools constraints-breeding. We observe those rats appearance weekly; detecting blood serum CORT, GAS, IL-2, blood plasma ACTH,MTL and weighing rats weight, spleen and thymus gland.
2 Compared with the Chaihushugan San group:the rats are divided into regular groups, model group, CHSGS group and SJZT Group by adopting Openfield test, ten each. The model group will be bred two weeks by keeping them alone and tools constraints. The regular group and model group will be physiological saline lavaged once a day. CHSGS group and SJZT group would be lavaged lml/100g once a day. Two weeks later, we observed each group on behavioral biology including Open field test and Forced Swimming test after the two hours later after the last lavage. Then we observe the actions of rats, use RAImmune to detect blood serum CORT、GAS. IL-2, blood plasm MTL; weigh body mass of rat and weight of spleen and thymic.
3 The Chemistry study of CHSGS group and liver-qi depression group ERK-CREB cell Signaling Pathway:we used the same way which as mentioned above to breed them, then killed the rats the day after two weeks. those model groups would be obtained two weeks later and observed brain organization Hippocampus japonicus CA1、CA3、DG and BLA section CREB. P-CREB. ERK1/2. P-ERK cell number and immune positive neuron.
Results:
1 Compared with the regular group, during making models, the rats are losing weights. There were big differences from week one to week four (P<0.001); blood serum decrease as well, and also the big differences between week two and week three (P<0.05) as well as week two to week four (P<0.05). Plasma ACTH and blood serum CORT increased between week two till week four. Blood serum and plasma increased at week three. Spleen and thymus gland decrease at week four (P<0.01,P<0.001) and immuno-cell factor IL-2 decrease at week four.
2 Compared with regular group, during making model, the model group increasing weight slowly and big differences (p<0.001); horizontal movement and vertical movement during of Open field test decrease (p<0.01), blood serum CORT and plasma ACTH increases (p＜0.001); plasma MTL and blood serum GAS decreases (P＜0.05); spleen weight and thymus gland weights decrease (p<0.01, p<0.001); blood serum IL-2 decrease a lot (p<0.01)
Compared with model group, CHSGS group increases a lot weight (p<0.001), so does the SJZT group (p＜0.001); horizontal score and vertical score of CHSGS group increases and the stay time in the central form and rats'shit decreased. SJZT group encountered the similar situation but make no differences (p>0.05); the immobility time of forced swimming of CHSGS group shortened and The SJZT group didn't and made no obvious differences (p>0.05); the amount of blood serum CORT and plasm ACTH from CHSGS group decreased (p<0.01, p＜0.001), the amount of blood serum CORT and plasm ACTH from The SJZT decreased a bit and made no obviously differences (p>0.05); the amount of blood serum GAS and plasm MTL from CHSGS group increased (p>0.05), however, the amount of blood serum GAS and plasm MTL from The SJZT group tend to increase as well, and made no obviously differences (p>0.05); the weight of spleen and thymus from both group increased (p<0.05, p<0.01);and blood serum IL-2 from group increased as well (p<0.05).
Compared with regular group, body weight from CHSGS group increased but with no differences (p>0.05), body weight from SJZT group increased slowly and with a lot different (p<0.001>); horizontal scores and vertical scores from both group increased, stay time in central form and shit amount decreased, but made no obviously differences (p>0.05); the immobility time of forced swimming from CHSGS group decreased obviously (p>0.05), made no obviously differences. The immobility time of forced swimming from The SJZT group shortened a lot and made great differences (p>0.001); the amount of blood serum CORT and plasma ACTH from both group decreased and made no obvious differences (p>0.05); the amount of blood serum GAS and plasma MTL from both group increased and made no obvious differences (p>0.05); the spleen weight and thymus gland weight from both group increased and made no obvious differences (p>0.05); the amount of blood serum IL-2 increased and made no obvious differences (P<0.05).
3 Compared with regular group, CA3, DG section from Hippocampus japonicus and BLA ERK1 amount of positive cell, the positive area from CA3 section ERK1 of Hippocampus japonicus decreased a lot. CA3, DG section from Hippocampus japonicus and BLA ERK2 amount of positive cell, the positive area from CA3 section ERK2 of Hippocampus japonicus decreased a lot; CA3, DG section and BLA P-ERK positive cell amount from Hippocampus japonicus, DG section and BLA positive area decreased a lot.; each section of Hippocampus japonicus from model group and BLA CREB, P-CREB amount of positive cell, the positive area decreased a lot.
Compared with model group, the Hippocampus japonicus CA3, DG section and BLA ERK1/2 amount of positive cell from CHSGS, and Hippocampus japonicus CA3 section ERK1/CA and BLA ERK2 increased obviously, and the same thing happen to the SJZT Group but made no obvious differences. Hippocampus japonicus CA3, DG section and amount number of BLAP-ERK positive cell, Hippocampus japonicus DG section and BLA positive area increased, those figures from The SJZT tend to increase as well but with no obvious differences;the amount of BLA CREB, P-CREB positive cell, positive area from each section of Hippocampus japonicus increased obviously, the same thing happened to The SJZT group but didn't make a lot differences.
Compared with regular group, the above figures from CHSGS group and SJZT group has no differences.
Conclusion:
1 This experiments focus on internal secretion, immunization, stomach and Intestines to observe the way to produce the different circumstances of stagnation of liver-QI on different period, which indicates that it only takes two weeks that can produce stagnation of liver-QI rats'model.
2 CHSGS can make scores by increasing horizontal movement and vertical movement of stagnation of liver-QI rats. It can improve the stagnation of liver-QI rats' behavior by extending stay in the central form, increasing the shit amount, shortening the forced swimming time.
3 CHSGS has the power of anti stagnation of liver-QI; it is probably related to improve the rats'blood ACTH and CORT, which restrains HPA axle. CHSGS can also adjust stomach and intestines by improving plasm MTL and GAS. It mainly improves immunization system by increasing the amount of stagnation of liver-QI rats'blood serum IL-2, spleen weight and thymus gland weight, from which improved the power of anti stagnation of liver-QI.
4 CHSGS has the power of anti stagnation of liver-QI; CHSGS can protect the damaged nerve cell; improve the brain functions, relieving stagnation of liver-QI rats' symptom by improving Hippocampus japonicus CA3, DG section and BLA ERK1/2, P-ERK signal pathway activities;it is probably related to the improvement each section of rats'Hippocampus japonicus and BLA section CREB, P-CREB. It boosts the recovery of those stressed damaged nerve cell, improves the stagnation of liver-QI rats'symptom, moreover, and improves the power of anti stagnation of liver-QI.
5 The way of relieving depression of the liver-qi can relieve depression of the liver-qi by improving the blood ACTH and CORT, which restrains HPA axle,improve the plasm MTL and GAS, improving immunization system by increasing the amount of stagnation of blood serum IL-2, spleen weight and thymus gland weight, improving Hippocampus japonicus CA3, DG section and BLA ERK1/2, P-ERK signal pathway activities, improving each section of Hippocampus japonicus and BLA section CREB, P-CREB.
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