Study On The Effect And Mechanism Of Yiqi Wenyang Huwei Decoction In Treating Bronchial Asthma Based On Cellular Autophagy MTOR Pathway

Chapter 1: Preparation and quality and main chemical composition identification of Yiqi Wenyang Huwei decoctionObjective: To identify the quality and main compound components of YWHD by Q-Orbitrap-LC-MS,so as to ensure the stability of the quality of the experimental drug.The chemical structure formulae and types of the main compound components of YWHD were also analyzed to reveal the main chemical substance bases and active molecular groups of YWHD to exert its medicinal effects.Methods: The volatile oils of Atractylodes macrocephala,Bifeng,Gui Zhi,Paeonia lactiflora and Ginger of YWHD herbal compound components were extracted with water and then decocted twice together with other remaining herbs,and the volatile oils were mixed with the concentrated solution and spray dried to obtain the dried paste powder.the dried paste powder samples of YWHD were processed,and the quality and chemical composition of YWHD were identified using Q-Orbitrap-LC-M,and the content of standards and chromatographic peaks were plotted.The standard curves were plotted against the peaks,and a Q Exactive high-resolution mass spectrometer equipped with an electrospray ionization source(ESI)was used for positive and negative ion switching scans.Full mass/dd-MS2 was used for qualitative detection and selected reaction monitoring(PRM)for quantitative detection.Results: A total of 5.6 kg of dried paste powder was extracted from 24 kg of YWHD raw herbs,with an extraction rate of 23.33%.The average content of peonidin in YWHD based on Q-Orbitrap-LC-MS method was 5824.807 μg/g;the average content of asclepiroside was 386.996 μg/g,glycyrrhizin was 563.932 μg/g,senecoside was61.570 μg/g,and 5-O-methylvisamitrin was 458.581 μg/g.458.581 μg/g,trans-cinnamaldehyde 1.826 ng/g,mullein isoflavone 122.602 μg/g,epimedioside38.485 μg/g,6-gingerol 100.381 μg/g,atractylenolide III 40.193 μg/g,8-gingerol The average contents of the YWHD extracts were 1384.877 ng/g,astragaloside 7998.865ng/g,glycyrrhetinic acid 819.767 μg/g,and 10-gingerol 400.246 ng/g.Based on QTOF identification,a total of 580 compounds were matched in mz Cloud for the components in YWHD extracts.A total of 281 compounds in the mz Cloud best match had a combined score greater than 60.Conclusion: The highest average content of peonidin in YWHD was 5824.807 μg/g by Q-Orbitrap-LC-MS method for quantitative analysis,while the qualitative analysis of compounds without standards showed that most of the components were mainly flavonoids,phenolic acids and terpenoids,etc.Chapter 2: Study on the effect and mechanism of YWHD intervention on asthmatic rats through regulating cellular autophagyObjective: The study mainly investigated the effect of YWHD on asthmatic rats based on the regulation of cellular autophagy by m TOR signaling and its mechanism of action.We established an asthma rat model by OVA induction and observed the effect of YWHD on the level of bronchial autophagy and the regulation of AMPK/PI3K/Akt/m TOR signaling pathway in lung tissues of bronchial asthma rats.Methods: SD male rats were randomly divided into normal control group,model group,YWHD low dose group,YWHD medium dose group,YWHD high dose group and dexamethasone group,8 rats in each group.The rats were injected intraperitoneally with 10% OVA+aluminum hydroxide suspension on day 0 and day 7,and nebulized with 2% OVA daily for 14 days from day 14,while the normal control group was given equal doses of saline.The rats in the positive control group were given 1 mg/kg of dexamethasone every 24 h.The rats in each group were discontinued 12 h before the last OVA nebulization on day 27.The rats were observed for general conditions and behavioral and lingual changes,and the changes in body weight were recorded.The levels of inflammatory factors IL4,IL13,IL6,IL33,IL25,TNF-α and Ig E in rat BALF were detected by Elisa.The levels of LC3II/LC3 I,p-PI3K/PI3 K,p-Akt/Akt,p-AMPK/AMPK and p-m TOR/m TOR were detected by western blot.Results: The rats in the model group showed sneezing,croup,nasal discharge,lazy movement,and a pale tongue,showing the overall symptoms of "weak qi-yang and deficient guard qi".elevated(P<0.05;P<0.01);reduced 6.25 mg/m L to 50 mg/m L;Mch-induced maximal airway resistance Max Rrs(P<0.01);reduced focal infiltration of lymphocytes and epithelial cupulocytosis and subepithelial collagen deposition in bronchial and perivascular lung tissue;reduced BALF eosinophil,neutrophil and lymphocyte counts(P<0.01;P<0.001);decreased the levels of BALF inflammatory factors IL4,IL13,IL6,IL33,IL25,TNF-α,Ig E(P<0.1;P<0.01;P<0.001);decreased the level of bronchial IL13 expression(P<0.01),and down-regulated lung tissue bronchial LC3 B,Beclin-1 expression levels(P<0.05;P<0.01;P<0.001),decreased the levels of autophagic vesicles as well as autophagic lysosomes in lung tissue cells(P<0.05,P<0.01),down-regulated lung tissue LC3II/LC3 I and p-AMPK/AMPK levels(P<0.01),and up-regulated p-m TOR/m TOR,p-PI3K/PI3 K,p Akt/Akt levels(P<0.05,P<0.01).Conclusion: YWHD inhibits excessive autophagy in airway epithelial cells through regulating AMPK/PI3K/Akt/m TOR signaling pathway,there by reducing the level of lung tissue and airway inflammation in asthmatic rats and improving lung tissue and airway pathological changes.Chapter 3: Study on the mechanism of action of YWHD-containing serum on autophagy of human bronchial epithelial cells 16HBEObjective: To investigate the effect of YWHD-containing serum on IL13-induced autophagy in human bronchial epithelial cells 16 HBE cells,based on AMPK/PI3K/Akt/m TOR signaling pathway using PI3 K inhibitor LY294002 and AMPK agonist Metformin treatment to verify the mechanism of action of YWHD-containing serum on 16 HBE autophagy.Methods: SPF male SD rats were randomly divided into control group,YWHD group and dexamethasone group,10 rats in each group;the YWHD group was given 12g-kg-1 daily,the dexamethasone group was given 1 mg-kg-1 daily,and the control group was given an equal volume of distilled water for 7 days;blood was collected 1h after the last administration to separate the serum.MTT method was used to detect the cytotoxicity of drug-containing serum and the levels of autophagic LC3II/LCI induced by IL13 in 16 HBE cells at 15 min,30min and 1h.m RFP-GFP-LC3 ADV transfection was performed to observe the effect of Yiqi-Wenyang Protective Tang drug-containing serum on the level of autophagic flow in 16 HBE cells induced by IL13,and the structure and number of autophagic vesicles and autophagic lysosomes in 16 HBE cells were detected by transmission electron microscopy.The structure and number of autophagic lysosomes were detected by transmission electron microscopy.p-PI3K/PI3 K,p-Akt/Akt,p-m TOR/m TOR,p-AMPK/AMPK,LC3II/LC3 I levels were detected by Western blot.p-PI3K/PI3 K,p-Akt/Akt,p-m TOR/m TOR,p-AMPK/AMPK,LC3II/LC3 I levels were detected by Western blot after treatment with PI3 K inhibitor LY294002 and AMPK agonist Metformin.blot to detect p-PI3K/PI3 K,p-Akt/Akt,p-m TOR/m TOR,p-AMPK/AMPK,LC3II/LC3 I levels to verify the mechanism of action of YWHD-containing serum on 16 HBE autophagy.Results: The percentage of YWHD-containing serum was greater than 15% acting on16 HBE cells for 48 hours to produce cytotoxicity(P<0.05;P<0.01);m RFP-GFP-LC3 ADV fluorescence double-labeling observed that YWHD-containing serum could reduce the mean fluorescence area of m RFP+GFP in IL13-induced 16 HBE cells(P<0.01).YWHD-containing serum decreased the mean number of autophagic vesicles and autophagic lysosomes in IL13-induced 16 HBE cells as observed by transmission electron microscopy(P<0.01).y WHD-containing serum decreased the mean autophagic vesicles and autophagic lysosomes in IL13-induced 16 HBE cells by upregulating p-PI3K/PI3 K,p-Akt/Akt and p-m TOR/m TOR levels,and downregulating p-AMPK/AMPK,Beclin1 and LC3II/LC3 I levels(P<0.05;P<0.01;P<0.001).After treatment of 16 HBE cells with PI3 K inhibitor LY294002 and AMPK agonist Metformin,respectively,p-PI3K/PI3 K,p-Akt/Akt and p-m TOR/m TOR levels were down-regulated and p-AMPK/AMPK,Beclin1 and LC3II/LC3 I levels were were upregulated(P<0.05;P<0.01;P<0.001).Conclusion: YWHD-containing serum inhibits IL13-induced excessive autophagy occurrence in human bronchial epithelial 16 HBE cells through multi-target and multi-pathway regulation of AMPK/PI3K/Akt/m TOR signaling pathway.Chapter 4: PF activates m TORC1 through competitive reversal of PRAS40-Raptor interaction and thereby inhibits 16 HBE cell autophagy Objective: To observe the intervention of paeoniflorin in m TORC1 activity regulatory subunit PRAS40 and Raptor interactions to inhibit 16 HBE cell autophagy.Based on m TORC1 activated by PRAS40-Raptor interactions,LV-h-PRAS40+ and PC-h-raptor(K207M&L302I&Q417H)treatments were used to validate paeoniflorin mechanism of action on 16 HBE autophagy.Methods: MTT method was used to screen the safe dose of PF action on 16 HBE cells;m RFP-GFP-LC3 ADV fluorescence traced the level of autophagic flow in 16 HBE cells;transmission electron microscopy was used to observe the structure and number of autophagic vesicles and autophagic lysosomes;Western blot was used to detect the effect of PF on IL13-induced autophagy in 16 HBE cells p-PI3K/PI3 K,p-Akt /Akt,p-m TOR/m TOR,p-AMPK/AMPK and LC3II/LC3 I levels;Autodock Tool to analyze the binding activity and sites of PF and m TORC1 activation subunit Raptor,Western blot to detect the effect of PF on IL13-induced autophagy of 16 HBE cells p-PRAS40/PRAS40,p-Raptor/Raptor levels;CO-IP assay of PF on IL13-induced autophagic PRAS40-Raptor interactions in 16 HBE cells;MTT assay to screen 16 HBE cells for resistance to puromycin concentrations and polybrene safe concentrations,LV-h-PRAS40+ effects safe MOI of 16 HBE cells;cellular immunofluorescence to detect the efficiency of LV-h-nc and PC-h-nc infection of 16 HBE cells and LV-h-PRAS40+ infection of 16 HBE cells with PRAS40 overexpression;Western blot to detect the effect of PF on IL13 induction of LV-h-PRAS40+ and PC-h-raptor(K207M&L302I&Q417H)16HBE cell autophagy p-m TOR/m TOR,p-Raptor/Raptor and p-PRAS40/PRAS40,LC3II/LC3 I levels;m RFP-GFP-LC3 ADV fluorescence tracing PF on IL13-induced LV-h-PRAS40+ and PC-h-raptor(K207M&L302I&Q417H)16HBE cells autophagic flow;transmission electron microscopy to observe the effect of PF on IL13-induced LV-h-PRAS40+ and PC-h-raptor(K207M&L302I&Q417H)16HBE cells autophagic vesicles and autophagic lysosome levels;CO-IP to detect Effect of PF on IL13-induced autophagic PRAS40-Raptor interactions between LV-h-PRAS40+ and PC-h-raptor(K207M&L302I&Q417H)16HBE cellsResults: PF reduced the mean fluorescence area of IL13-induced m RFP+GFP in16 HBE cells,inhibited the level of autophagic flow(P<0.01;P<0.001),decreased the mean number of autophagic vesicles and autophagic lysosomes in IL13-induced16 HBE cells(P<0.01;P<0.001),upregulated the level of p-m TOR/m TOR,and decreased the levels of LC3II/LC3I(P<0.05;P<0.01;P<0.001),but did not significantly alter p-PI3K/PI3 K,p-Akt/Akt and p-AMPK/AMPK(P>0.05),PF upregulated IL13-induced p-PRAS40/PRAS40 levels after autophagy in 16 HBE cells,and decreased the level of p-Raptor/Raptor(P<0.01;P<0.001).PF decreased IL13-induced PRAS40-Raptor interactions and elevated Raptor-m TOR interactions after autophagy in 16 HBE cells.After LV-h-PRAS40+ and PC-h-raptor(K207M&L302I&Q417H)treatment of 16 HBE cells increased IL13-induced m RFP+GFP mean fluorescence area of 16 HBE cells compared with PF+IL13(P<0.01;P<0.001),elevated IL13-induced 16 HBE cells mean autophagic vesicle and autophagic lysosome numbers,decreased p-m TOR/m TOR levels and upregulated LC3II/LC3 I levels,decreased p-PRAS40/PRAS40 levels and elevated p-Raptor/Raptor levels,and elevated IL13-induced PRAS40-Raptor interactions after autophagy in 16 HBE cells,and decreased Raptor-m TOR interactions(P<0.05;P<0.01;P<0.001).Conclusion: PF inhibits IL13-induced excessive autophagy in human bronchial epithelial 16 HBE cells by competitively reversing the activation of m TORC1 through the interaction between PRAS40 and Raptor.