| Objective Idiopathic pulmonary fibrosis(IPF)is a kind of interstitial pneumonia characterized by injury,inflammation,immunity and fibrous deposition.Traditional Chinese medicine classifies it as lung arthralgia and lung flaccidity,we sorted out and synthesized the related traditional Chinese medical treatises of the representative doctors of the past dynasties,clarified the pathogenesis of the disease in traditional Chinese medicine and the commonly used treatment methods and medicines,and highlights the important role of the method of Peitushengjin Tongluo Method.At the same time,based on the theoretical and experimental research foundation of Chinese and Western medicine in the early stage of the research group,the model of IPF mice with deficiency of lung and spleen and the model of macrophage induced by lipopolysaccharide(LPS)were copied,and the deposition of IPF development was regulated from the perspective of NLRP3 inflammasome mediated pyroptosis,and the representative formula of "Shenlingbaizhu powder" and "Maimendong Decoction" for Peitushengjin Tongluo Method could be regulated by regulating the high surface of NF-κbp65.It can provide a reliable theoretical,clinical and experimental basis for the treatment of IPF.Method:1 Theoretical study:refer to the relevant ancient literature and modern research reports of lung arthralgia,lung flaccidity,IPF,Peitu Shengjin Tongluo recorded by ancient and modern Chinese physicians,analyze the common pathogenesis characteristics and treatment methods of lung arthralgia,lung flaccidity and IPF,and clarify the TCM Mechanism of Peitu Sheng Jin Tongluo method in the prevention and treatment of IPF.2 Experimental study:The animal experiment used C57BL/6 mice as the IPF model of lung and spleen deficiency,and LPS induced macrophage model for cell experiment.In the animal experiment,the model of IPF with deficiency of lung and spleen was established firstly,and the syndrome of spleen deficiency was evaluated by the observation of body weight and the apparent condition of spleen deficiency syndrome;the influence of "Shenlingbaizhu powder" and "Maimendong Decoction" on the model of IPF was judged by the observation of general state,HE staining of lung tissue and pathological grading standard of Masson staining.The effects of "Shenling baizhu powder" and "Maimendong Decoction" on IL-1β,IL-18 and TGF-β1 in plasma of mice with IPF deficiency of lung and spleen were detected by ELISA.The effect of "Shenling baizhu powder" and "Maimendong Decoction" on the expression of collagenⅢ,NLRP3,caspase-1,NF-κbp65protein in lung tissue of IPF model mice with deficiency of lung and spleen was detected by Western blot.And the effect of it the expression of NLRP3,caspase-1,IL-1β,IL-18 m RNA in lung tissue of IPF mice with deficiency of lung and spleen was detected by RT-PCR.In the cell experiment,the macrophage model induced by lipopolysaccharide(LPS)was established firstly.After the intervention of the serum containing medicine of "Shenlingbaizhu powder" and "Maimendong Decoction",the influence of the serum containing medicine on IL-1β and IL-18 in the culture medium of LPS induced macrophage model was detected by ELISA,and the protein NLRP3 and caspase-1 was detected by immunofluorescence.Results:1.Animal experiment results1.1 observation results of spleen deficiency model in mice1.1.1 comparison of body weight of mice in each group before and after the model of spleen deficiency syndrome During the experiment,the body weight was measured three times before the establishment of the model of spleen deficiency,the fourth day and the seventh day.It was found that the body weight of the blank control group and the sham operation control group increased slowly,but there was no statistical difference between the three times(P>0.05).There was no statistical difference between the body weight of the model of spleen deficiency before the establishment of the model of spleen deficiency,the normal group and the sham operation group(P> 0.05).There were significant differences in body weight before the establishment of spleen deficiency model,on the 4th day and the 7th day(P<0.05).The results showed that the weight decreased in turn,the lowest on the 7th day compared with the other two time periods,the second on the 4th day,and the highest before the establishment of spleen deficiency model(P<0.05).1.1.2 observation on general state of mice with spleen deficiency syndrome the grading statistics of the apparent situation of mice in each group showed that:compared with the blank control group,the grading scores of five kinds of spleen deficiency model in body odor,posture,body heat and cold,hair and feces were significantly higher than those in the blank control group(P<0.05),and there was no statistical difference between the sham operation control group and the blank control group(P>0.05).1.2 The observation results of mice IPF model of lung and spleen deficiency1.2.1 observation of general state After the model of pulmonary fibrosis(lung deficiency)was established on the basis of the spleen deficiency model,it was found that the mice in each group showed shortness of breath,cough and scratching nose,panting sound in the throat,depression and fatigue after the model was established Languor,curl up,slow response,hair haggard and easy to fall off,poor appetite,less water,and so on.However,the blank control group and sham operation control group had no presentation.After 28 days of treatment,it was found that the general state of mice in hormone control group and three Chinese medicine dose groups were better than those in model group,such as spirit,quick response,hair color,occasional cough,Shortness of breath,poor food intake and so on;The general state of the mice in hormone control group and high and medium dose group was better than that in low dose group.1.2.2 pathological results of lung tissue1.2.2.1 HE staining results In the blank control group,the lung tissues such as bronchi,alveolar ducts,alveolar sacs,alveoli and alveolar septum were intact.The lung tissue structure of the sham operated control group was similar to that of the blank control group.In the model control group,there were structural disorder,alveolar collapse and fusion,alveolar wall thickening,inflammatory cell infiltration,fibroblast proliferation,fibrous tissue deposition,large consolidation area.In the hormone control group,the low-dose group,the middle dose group and the high-dose group,the structure of lung tissue was disordered,but the degree was less than that of the model control group,and the damage was more serious than that of the blank control group and the sham operation control group.HE staining showed that:Compared with the blank control group,the pathological scores of lung tissue in the model control group and the four drug treatment groups were significantly higher(P<0.01),but there was no significant difference between the sham operation control group and the blank control group(P>0.05);Compared with the model control group,Except for the low dose group,the other groups of mice lung pathological scores were significantly reduced(P<0.05),while the low dose group had no significant difference(P>0.05).1.2.2.2 Masson staining results In the blank control group,the structure of bronchi,alveoli and other lung tissues was complete,and a small amount of blue collagen fibers were occasionally seen on the alveolar septum.The lung tissue structure of the sham operated control group was similar to that of the blank control group.But the structure was disordered and fibrosis was observed In the model control group.In the area of flaky mutation,there were obvious blue collagen fibers in alveolar septum,bronchi and vascular models.In the hormone control group,the low-dose group,the middle dose group and the high-dose group,the structure of lung tissue was disordered,the degree of fibrosis was lighter than that in the model control group,and heavier than that in the blank control group and the sham operation control group.Mild,moderate and severe pulmonary fibrosis were found in each group.The score of collagen increment in lung tissue in Masson staining showed that:Compared with the blank control group,The level of collagen in lung tissue of mice in the model control group and four drug treatment groups was significantly higher(P<0.01),but there was no significant difference in the sham operation control group(P>0.05);On the contrary,the pathological scores of other groups were significantly reduced to the model control group except for low dose group of traditional Chinese Medicine(P<0.05),while it had no significant difference(P> 0.05).1.3 ELISA test results IL-1β,IL-18 and TGF-β1 in plasma of mice in the model control group and four drug treatment groups were extremelysignificantly increased to the blank control group(P<0.01),but there was no significant difference in the sham operation control group(P>0.05);On the contrary,the levels of them in other groups were significantly reduced to the model control group(P<0.05).The levels of them in hormone control group,middle dose group and high dose group were significantly declined to those in low dose group(P<0.05).1.4 Western blot test results In contrast with the blank control group,the lung tissue collagenⅢ,NLRP3,caspase-1 and NF-kβp65 expression in the model control group and four drug treatment groups were significantly increased(P<0.01),but there was no significant difference in the sham operation control group(P>0.05).On the contrary,the expression of them in other groups were significantly reduced to the model control group(P<0.05).The expression of them in the middle dose group and high dose group were significantly lower than those in the low dose group(P<0.05).It has no significant difference in NLRP3,caspase-1 and NF-k β p65 expression between the hormone control group and the middle dose group(P>0.05).There was no significant difference in collagenⅢ、NLRP3 and Caspase-1 expression between middle dose group and high dose group(P>0.05).1.5 RT-PCR experiment results In contrast with the blank control group,IL-1β,IL-18,NLRP3,caspase-1,NF-k β p65 m RNA expression in other groups were significantly higher except for sham operation control group(P<0.01),but there was no significant difference in the sham operation control group(P>0.05);But the expression of them in other groups decreased significantly to the model control group(P<0.05).The expression of them in hormone control group,middle and high two dose groups were significantly lower than low dose group(P<0.05).The level of IL-18 m RNA in high dose group was significantly lower than that in hormone control group and middle dose group(P<0.05).It has no significant difference in them between the middle and high two dose groups(P>0.05).2 Cell test results According to the statistics of IL-1β and IL-18 content in supernatant of cell culture at 24 hours,the content of them in other groups extremely significantly increased in contrast with the blank control group except for normal serum control group(P<0.01),while it has no significant difference between normal serum control group and blank control group(P>0.05).The contents of them in other groups were significantly reduced in contrast with the model control group(P<0.05).The contents of them in the middle dose group,high dose group and NF-κB inhibition control group were significantly lower than those in the low dose group(P<0.05).IL-1βcontent has no significant difference of between high dose group and NF-κB inhibition control group(P>0.05).The results of the mean fluorescence intensity of NLRP3 and caspase-1 in each group was examined at 24 hours showed that the average fluorescence intensity of their protein in other groups increased significantly in contrast with the blank control group expect for normal serum control group(P<0.01),but it has no significant difference between normal serum control group and blank control group(P>0.05).Compared with the model control group,the average fluorescence intensity of their protein in other groups were significantly decreased(P<0.05).The average fluorescence intensity of their protein in middle dose group,high dose group and NF-κB inhibition control group were significantly lower than that in low dose group(P<0.05).There was no significant difference between high dose group and NF-κB inhibition control group(P>0.05).Conclusion1 From the theoretical discussion,we find that the pathogenes is of IPF is mainly deficiency of lung qi and Yin,accompanied by deficiency of spleen and kidney.In addition,phlegm,blood stasis and collaterals are mixed with each other.Peitushengjintongluo method is a common method to treat IPF.2 The IPF mouse combination model of spleen deficiency and lung deficiency can show the similar syndrome of IPF lung and spleen deficiency,which is similar to IPF lung and spleen deficiency syndrome complex model of rats.3 Shenlingbaizhu powder and Maimendong Decoction can improve IPF by regulating NLRP3 related gene protein.4 The effect of Shenling baizhu powder and Maimendong Decoction on NLRP3 nflammasome is related to the expression of NF-κ B regulated,which may be one of the mechanisms of this method. |
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