| Objective1 To explore the functional characteristics of the viscera in the stable stage of chronic obstructive pulmonary disease(COPD)from the perspectives of the material basis of respiratory function,the source of viscera function and the relationship between cold and heat may provide theoretical basis for subsequent clinical and experimental research.2 To analyze the distribution of clinical symptoms and signs and the essence of pathogenesis in patients with stable COPD and to deepen the understanding of the functional characteristics of the viscera.3 We explore and establish a TCM disease-symptom combination animal model that reflects the pathogenesis of stable COPD,observe the macroscopic and microscopic features of the model,and initially establish the model evaluation method by the carrier of animals.4 This study observes the curative effect of Recuperating lung decoction(RLD)on stable COPD mouse model by measuring multiple indicators and illustrates that RLD may regulate the stable phase of COPD through TLR4-PI3K-Akt-mTOR pathway,which is a key way to achieve the inhibition of inflammatory response.Methods1 By analyzing the related theories and landmark achievements of traditional Chinese medicine on the stable COPD stage and its influence on the eight principles of syndrome differentiation and treatment,we will explore the pathogenesis and zang-fu function characteristics that may be more in line with the clinical reality of the disease.2 The observational research method was used to uniformly input the basic data,main symptoms,concurrent symptoms,tongue and pulse,etc.of the included patients with stable COPD.The distribution of symptoms and signs in stable COPD was mined through component ratio analysis,cluster analysis and factor analysis to explain the zang-fu function characteristics.3 In the exploratory study,firstly,according to the possible influencing factors of deficiency of the lung and spleen core two organs,choose senna decoction,fasting on alternate days,noise stimulation and hydrocortisone.Then compare single,two and multiple intervention factors to determine the optimal modeling method of the lung and spleen core two multi-organ deficiency model.Secondly,combined with smoke exposure(CS)and lipopolysaccharide(LPS)intervention.Thirdly,Combining the clinical understanding of the multiple viscera deficiency in the core of the lung and spleen,as well as the general condition of the mice after modeling,mortality,lung function and the degree of compliance with the etiology and other factors,the modeling method of stable COPD of multiple viscera deficiency in the core of the lung and spleen was initially determined.4 In the formal experiment,90 BALB/c mice were divided into 20 blank control group,10 simple COPD stable stage group,and 60 COPD stable stage group with multiple viscera deficiency in the core of the lung and spleen.According to the method finally established in the preliminary experiment to establish a stable COPD model with lung and spleen core two multiple viscera deficiency.Randomly select 10 mice in each group to test the general condition,lung tissue pathology and lung function to determine the stability and repeatability of the experiment.And then explore their possible microscopic characteristics of D-xylase,somatostatin,cortisol,trans-triiodothyronine,testosterone and estradiol expression levels.5 The 50 mice in the stable COPD group with multiple viscera deficiency in the core of the lung and spleen were randomly divided into model group,positive drug group,RLD low-dose group,medium-dose group and the high-dose group,the other 10 were blank control groups.After 28 days of drug administration and intervention,the general conditions of the mice were evaluated,such as body weight,rectal temperature,water volume,lung function,etc..Experimental specimens such as lung tissue,serum and BALF were prepared,and the enzymelinked immunosorbent assay(ELISA)method was used to detect inflammatory factors,including IL-6,IL-8,IL-10,TNF-α,IL-1β and macrophage inflammatory protein 2(MIP-2)and oxidative stress indicator,such as 8-hydroxydeoxyguanosine(8-OHdG)and total antioxidant capacity(T-AOC).Then the expression levels of TLR4-PI3K-Akt-mTOR protein and mRNA were detected by Rt-PCR and WB technology to explore the curative effect and possible mechanism of RLD on stable COPD model mice.Results1 Multiple viscera deficiency in the core of the lung and spleen reflect the complex functional relationship of the viscera in COPD,which requires a correct understanding of COPD stable specimens and the relationship between cold and heat.The lung and spleen as the core aims to emphasize the material basis of the respiratory function of the lungs in TCM and the latter are intended to focus on the source of the functions of the zang-fu organs.Therefore,the functional positioning of the multiple viscera deficiency in the core of the lung and spleen has the advanced nature of matching the clinical reality of COPD in stable stage.2 A total of 231 patients with stable COPD were included.The composition ratio analysis showed that the main symptoms of the included patients were cough,wheezing,shortness of breath,and chest tightness in descending order.The other symptoms are night frequency,chills and cold limbs,dry mouth,difficulty falling asleep,light sleep and easy awakening,spontaneous perspiration,tinnitus,fatigue,loss of appetite,abdominal distension,etc..The tongue color is mainly dark red and the tongue coating is mainly thin white coating,followed by white greasy coating and thin yellow moss.The pulse condition is mainly thin veins.Factor analysis obtained 14 common factors through factor rotation of 35 symptom variables.The symptom variables included were divided the clinical symptoms and signs of stable COPD into three categories by R-type hierarchical clustering,which more objectively reflected the zangfu functional characteristics of stable COPD and the pathological characteristics of the deficiency and excess and the mixed cold and heat.3 In this study,a mouse model of COPD with multiple viscera deficiency in the core of the lung and spleen was successfully established in the stable period.3.1 We comprehensively detect body weight,water intake,rectal temperature,symptom total score,lung function and the degree of compliance with the cause of each group of mice after the end of the syndrome and 2,4,8,and 12 weeks of smoke exposure(CS).It was found that the combined application of hydrocortisone,hunger and satiety,noise stimulation,senna,CS and LPS was the best method for establishing the COPD model with multiple viscera deficiency in the core of the lung and spleen.3.2 In the formal experiment,it was found that the body weight,food intake and rectal temperature of the COPD group with multiple viscera deficiency in the core of the lung and spleen and the simple COPD model group were lower than those of the blank control group,and the water intake and total symptom score were higher than those of the blank control group.Compared with the simple COPD model group,the rectal temperature in the group decreased significantly(P<0.01),and the total symptom score increased(P<0.05).This indicated that the mice in the COPD group of multiple viscera deficiency in the core of the lung and spleen had more obvious visceral dysfunction.Lung histopathology showed that compared with the normal control group,the alveolar wall became thinner or ruptured,the alveolar cavity was enlarged and there were inflammatory cell infiltration around the airway in the COPD group with multiple viscera deficiency in the core of the lung and spleen and the simple COPD model group.The inflammatory infiltration,basement membrane edema and intraluminal exudation were more severe in the COPD group with multiple viscera deficiency in the core of the lung and spleen.Through ELISA to explore the microscopic characteristics of the model,it was found that the somatostatin in the COPD group with multiple viscera deficiency in the core of the lung and spleen was higher than that in the blank control group(P<0.01),and the levels of D-xylose,gastrin,estradiol and cortisol were lower than those in the blank control group(P<0.01 or P<0.05).The levels of transtriiodothyronine and testosterone were lower than those in the blank control group,but there was no difference(P>0.05).4 RLD can partially relieve the general state,lung function,lung tissue pathology,inflammatory injury and oxidative stress state of COPD mice with multiple viscera deficiency in the core of the lung and spleen and improve respiratory function.4.1 General status:The body weight,food amount,and rectal temperature of the two COPD model of multiple viscera deficiency in the core of the lung and spleen group were significantly lower than the blank control group(P<0.05 or P<0.01),while the total symptom score was significantly increased(P<0.01).The general state of the positive drug group and the RLD middle and high dose groups were improved to varying degrees after administration and treatment.Among them,the body weight,food amount and water intake increased,but there was no statistical difference(P>0.05).The rectal temperature of each administration group was significantly improved compared with the model group(P<0.01).The RLD lowdose group and the positive drug group were significantly improved(P<0.01).Although the total symptom score of the RLD group was lower than that of the model group,there was no statistical difference(P>0.05).It is suggested that the RLD middle and high dose groups have a good effect on improving the macroscopic performance of mice,which indicates that the RLD middle and high dose groups have certain advantages in regulating the state of viscera function deficiency.4.2 Pulmonary function:Ers,PEF,FEV0.1/FVC,FEV0.05/FVC,FEV0.2/FVC of the mice in the COPD model group of multiple viscera deficiency in the core of the lung and spleen were lower than those in the blank control group,while the Rrs,Crs,IC,FVC,FEV0.1,FEV0.05,and FEV0.2 of mice were higher than those in the blank control group.It indicates that the model mice have increased airway resistance and the possibility of small airway obstruction.The pulmonary function of each administration group was improved after administration.The improvement was more obvious in the positive drug group and the RLD high-dose group,followed by the middle-dose group.Therefore RLD can improve the airway elasticity to a certain extent and reduce the airway resistance and the degree of obstruction in the model mice.4.3 Pathology of lung tissue:Compared with the blank control group,the airways of the mice in the COPD model group were in a state of hypersecretion,inflammatory cells infiltrated in the alveoli,enlarged alveolar cavity and small airway epithelial hyperplasia or edema,etc..Compared with the model group,the above pathological conditions of the mice in each administration group were relieved to varying degrees.The RLD middle and high dose groups and the positive drug group were more obvious.4.4 BALF inflammatory cell count:The numbers of neutrophils,lymphocytes,and macrophages in the B ALF of the mice in the COPD model group of multiple viscera deficiency in the core of the lung and spleen were higher than those in the blank control group(P<0.05 or P<0.01).The number of lymphocytes in the RLD middle and high dose groups and the positive drug group was significantly lower than that in the model group(P<0.05 or P<0.01).The numbers of neutrophils and phages in the RLD middle and high dose groups were lower than those in the model group,but there was no difference(P>0.05).4.5 Anti-inflammatory injury and oxidative stress:The levels of IL-6,IL-8,IL-10,IL-1β,TNF-α and MIP-2 in the COPD model group of multiple viscera deficiency in the core of the lung and spleen were significantly higher than those in the blank control group(P<0.01).The levels of these indicators in the positive drug group and the RLD high-dose group decreased more significantly after treatment(P<0.05 or P<0.01),followed by the middle-dose group which always better than the model group(P<0.05 or P<0.01).Compared with the blank control group,the 8-OHdG level in the COPD model group of multiple viscera deficiency in the core of the lung and spleen was significantly higher(P<0.01).The levels of 8-OHdG in each administration group decreased after treatment(P<0.05 or P<0.01).And the RLD highdose group had the best effect,followed by the RLD medium-dose group.The T-AOC level of the mice in the COPD model group of multiple viscera deficiency in the core of the lung and spleen was lower than that in the blank control group.There were different degrees of elevation,but no difference was found,which suggests that RLD may reduce oxidative stress injury mainly by inhibiting the level of oxidative stress.5 RLD through the TLR4-PI3K-Akt-mTOR pathway may be the key way to regulate the stable phase of COPD to suppress the inflammatory response.Compared with the blank control group,the expression levels of TLR4,PI3K,Akt,mTOR mRNA and TLR4/GAPDH、pPI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR protein in the COPD model group with multiple viscera deficiency in the core of the lung and spleen were significantly higher(P<0.01).After intervention,all administration groups decreased after treatment,especially the positive group and RLD middle and high dose groups.It shows that RLD may inhibit the stimulation of PI3KAkt-mTOR signaling pathway by reducing the expression level of TLR4 protein in the lung tissue of model mice to reduce lung inflammation and improving respiratory function.Conclusion1 Multiple viscera deficiency in the core of the lung and spleen reflects the complex functional relationship of the viscera during the stable COPD period.The lung and spleen as the core aims to emphasize the material basis of the TCM lung governing qi and respiratory function and the two viscera pay attention to the source of the functions of the zang-fu organs.2 Patients in the stable stage of clinical COPD are characterized by coexistence of deficiency and excess,mixed with cold and heat,which shows the functional characteristics of the multiple viscera deficiency in the core of the lung and spleen.3 Combined application of senna,hunger and satiety,noise stimulation,hydrocortisone,CS and LPS compound methods can establish stable COPD models with multiple viscera deficiency in the core of the lung and spleen that reflect the clinical practice of traditional Chinese medicine and reflect the overall functional relationship of the viscera.Model evaluation must be comprehensively judged by general conditions,lung tissue pathology,lung function and microscopic indicators of syndromes.4 RLD has good curative effect in the treatment of COPD with multiple viscera deficiency in stable stage.RLD may reduce the pulmonary inflammatory response and improve respiratory function by downregulating the expression level of TLR4 protein and inhibiting the activation of the downstream PI3K-Akt-mTOR signaling pathway. |
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