| [Background]Normal and orderly water metabolism is an important part of maintaining human life activities.Abnormal water metabolism can produce pathological products such as water,fluid and phlegm."Fluid" and "Phlegm" have been confused in the academic history of TCM.Ancient Chinese medical doctors have mostly respected Zhang Zhongjing’s works on the discussion of fluid disease.However,Zhang Zhongjing’s description of "Fluid and Phlegm"actually refers to the fluid.Xiaoqinglong Decoction is a famous classic prescription for dispersing fluid and it has a rigorous formula.It can be divided into four Fang-Yuan.The first is Guizhi-Gancao Decoction of warming Yang,the second is Shaoyao-Gancao Decoction of nourishing Yin.The third is the Jiang-Xin-Wei Decoction which is commonly used in the therapy of nourishing both lung and spleen,relieving cough and arresting diarrhea.The fourth is the Banxia-Mahuang Decoction which is intended for the treatment of both exterior and interior and is longer than relieving asthma and palpitation.The four Fang-Yuan involve the theories Yin and Yang,viscera,Exterior and Interior.And their ultimate function is to regulate the metabolism of the body fluid.The pathogenesis of water retention in traditional Chinese medicine is closely related to the diseases of multiple systems in modern medicine.Therefore,it is of great significance to explore the composition principles and mechanism of Xiaoqinglong Decoction from the perspective of Fang-Yuan.[Objective](1)Eablishment and evaluation of the model of syndrome of accumulation of cold fluid in the lung based on the pathogenesis principle of "coldness of body and drinking cold tend to impair lung";(2)Preliminary study on the regulation of Xiaoqinglong Decoction and its Fang-Yuan on lung fluid transport based on the model of syndrome of accumulation of cold fluid in the lung.[Methods](1)The model of syndrome of accumulation of cold fluid in the lung was established by the modeling method of "coldness of body+drinking cold + cold bath".Through macroscopic comparison of rats’ general morphology,histopathology(HE and AB-PAS)and pulmonary function tests,ELISA quantitative analysis of inflammation indicators TNF-α,cAMP、cGMP in lung tissues of rats,immunohistochemistry method to determine the expression of AQP5 and MUC5AC,to determine whether the model is successful;(2)Xiaoqinglong Decoction and its Fang-Yuan were used to intervene the model rats.The general appearance and behavior,pathological morphology(HE and AB-PAS)and pulmonary function parameters were observed;The quantity of TNF-α,cAMP and cGMP in lung tissues of rats,Ghrelin and Obestatin in serum of rats were detected by ELISA;The expressions of AQP1,AQP5,MUC5AC,α-ENaC and Na+-K+-ATPase in lung tissues were detected by immunohistochemistry;The mRNA expressions of cAMP,PKA and CREB in lung tissues were detected by RT-qPCR and Western Blot was used to detect the expression of cAMP,PKA,CREB and p-CREB proteins.[Results](1)The experimental results showed that compared with the control group,the rats in the model group showed similar performance to those in the clinical patients with syndrome of accumulation of cold fluid in the lung,including symptoms and signs such as weight loss,increase of oral and nasal secretions,less movement,reduction of diet,and obvious inhibition of respiratory function.Xiaoqinglong Decoction and its Fang-Yuan could improve the abnormal state of model rats to various degrees;(2)Pathomorphological observation of HE staining showed that a large number of inflammatory cells gathered in the lungs of rats in the model group,the alveolar cavity was filled with edema,and the surrounding tissues were hyperemia.Xiaoqinglong Decoction and its Fang-Yuan could alleviate the pathological changes of lung tissues in the model rats to varying degrees;(3)Pathomorphological observation of AB-PAS staining observed that there was a large number of goblet cells in the bronchial epithelium of the lung tissues in the model rats,Xiaoqinglong Decoction and its Fang-Yuan could inhibit the metaplasia of goblet cells to different degrees;(4)Compared with the rats in control group,pulmonary function parameters of FEF50,TV,MMEF,FVC,IC and ERV of the model rats were significantly decreased.Both tI and tE were prolonged,and the differences were statistically significant(P<0.01).Xiaoqinglong Decoction and its Fang-Yuan could improve the pulmonary function of model rats to varying degrees;(5)Compared with the rats in control group,the cAMP in the lung tissues of the model rats was significantly reduced(P<0.01),the cGMP was increased(P<0.01),and the ratio was also significantly decreased(P<0.05),and the differences were statistically significant.Xiaoqinglong Decoction could markedly reverse these changes(P<0.01).After the intervention of every Fang-Yuan,compared with the model rats,the expression of CAMP was increased(P<0.01),along with decreased expression of cGMP(P<0.01).Taking cAMP/cGMP as evaluation index,the intervention effects of every Fang-Yuan were as follows:Xiaoqinglong Decoction>Jiang-Xin-Wei Decoction>Banxia-Mahuang Decoction>Guizhi-Gancao Decoction>Shaoyao-Gancao Decoction;(6)Compared with the rats in control group,the expression of TNF-α in lung tissue of model rats was up-regulated with statistical significance(P<0.01).After the intervention of Xiaoqinglong Decoction and every Fang-Yuan,the expression of TNF-α showed a downward trend to different degrees;(7)Compared with the rats in control group,the expression of AQP5 and α-ENaC(P<0.05)in the lung tissues of model rats were decreased,while the expression of MUC5AC was increased,and the difference was statistically significant(P<0.05).Xiaoqinglong Decoction and its Fang-Yuan can increase the positive expression of AQP5 and α-ENaC,and inhibit the expression of MUC5AC.There was no significant difference in AQP1 and Na+-K+-ATPase between each group,but Xiaoqinglong Decoction and its Fang-Yuan could numerically increase the expression of AQP1;(8)Compared with the rats in control group,the expression of Ghrelin and Obestatin in the serum of model rats were significantly decreased(P<0.01),and the ratio was also significantly decreased(P<0.01)with statistical significance.Taking Ghrelin/Obstatin as evaluation index,the intervention effects of every Fang-Yuan were as follows:Jiang-Xin-Wei Decoction>Xiaoqinglong Decoction>Guizhi-Gancao Decoction>Banxia-Mahuang Decoction>Shaoyao-Gancao Decoction;(9)Compared with the rats in control group,the mRNA and protein expression of cAMP,PKA and CREB in the lung tissues of the model rats were significantly inhibited(P<0.01)with statistical significance,the expression of p-CREB was decreased(P>0.05).Xiaoqinglong Decoction and its Fang-Yuan can up-regulate cAMP,PKA,CREB mRNA and cAMP,PKA,CREB,and p-CREB protein expression to varying degrees.[Conclusion](1)This model was consistent with the manifestation of syndrome of accumulation of cold fluid in lung from the aspects of general appearance and behavior,pulmonary function parameters and pathomorphological observation of rats.From the detection of laboratory indexes,the decrease of cAMP/cGMP expression was used to evaluate the change of lung-yang deficiency,and the decrease of AQP5 expression combined with MUC5AC over-expression was used to evaluate the degree of water retention.At the same time,Xiaoqinglong Decoction,as a classic prescription of dispersing cold and resolving fluid retention,its therapeutic effect can also be used as evidence for the success of model establishment.Therefore,from the perspective of macro-function to micro-molecular expression,this model is more in line with the pathological characteristics of syndrome of accumulation of cold fluid in the lung in TCM.Through the modeling method of "coldness of body+drinking cold+cold bath",the pathogenesis of syndrome of accumulation of cold fluid in the lung can be reproduced,and it has certain reliability and feasibility;(2)Based on the model of syndrome of accumulation of cold fluid in the lung,the expression of cAMP/cGMP in every group was observed.It was considered that GuizhiGancao Fangyuan and Shaoyao-Gancao Fangyuan may play the roles of warming yang and nourishing yin,respectively,leading to the role of yang-qi and yin-qi in the regulation of water metabolism.cAMP/cGMP may also be involved in the process of Banxia-Mahuang Fangyuan playing its role in relieving asthma and cough and dispersing cold and resolving fluid retention;(3)Ghrelin and Obstatin as a pair of indicators related to the body’s energy metabolism,Jiang-Xin-Wei Fang-Yuan may play its role of lung and spleen by regulating the content of the two,regulating the water channel and transporting water.(4)The expression changes of cAMP/PKA signaling pathway and lung water transportrelated proteins(AQP 1、AQP5、α-ENaC and Na+-K+-ATPase)before and after the intervention were observed.It was considered that Xiaoqinglong Decoction and its prescription may regulate the expression of some genes and proteins in lung tissue of rats through cAMP/PKA signaling pathway,so as to improve the pathological state of lung tissue,regulate lung water transport,promote the recovery of lung fluid balance,and have a certain protective effect on the lung of rats with syndrome of accumulation of cold fluid in the lung. |
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