Research On Mechanism Of Shenqi Huatan Formula In The Treatment Of Copd Based On MTOR Signal Pathway Regulating Autophagy

Objective Taking m TOR signal pathway as the breakthrough point,observe the effect of Shenqi Huatan recipe(SQHT)on human bronchial epithelial cells(16HBE)and autophagy in COPD model rats,and explore the molecular mechanism of Shenqi Huatan recipe in the treatment of chronic obstructive pulmonary disease(COPD).MethodsChapter 1: Effect of m TOR gene silencing on autophagy of 16 HBE cells16HBE cells were cultured in vitro;Prepare cigarette smoking extract(CSE),and select the best action time and concentration by CCK-8 method to pretreat the cells;The m TOR small RNA interference sequence was constructed,the silencing effect was detected by q RT-PCR,and the best interference sequence was selected.After transfection,the cells were divided into blank control group(BC group),model group(M group),m TOR si RNA transfection group(si-m TOR group),transfection control group(si-NC group),m TOR kinase activator 3BDO group(3BDO group).After treatment,the cells in each group were detected as follows: the proliferation ability of cells was detected by CCK-8,the migration ability of cells was detected by Transwell,and the apoptosis rate was detected by FCM;The formation of autophagosome was observed by MDC staining;The expression levels of LC3 B and Beclin-1 protein were detected by if method;The expression levels of PI3 K,AKT,m TOR,p62,LC3-Ⅱ/LC3-Ⅰ and Beclin-1 were detected by WB and q RT-PCR.Chapter 2: regulation of Shenqi Huatan Formula on autophagy of 16 HBE cells20 SD rats were randomly divided into two groups to prepare SQHT medicated serum and blank serum;CCK-8 method was used to detect the effect of SQHT containing serum on the activity of 16 HBE cells,and the best action time and concentration were selected for the next experiment;The cells in each group were intervened with medium containing 2.5% CSE.After 48 hours of culture,they were divided into control group(group A),model group(group B),SQHT containing serum group(Group C),rapamycin group(Group D),3-m A group(Group E),rapamycin + SQHT containing serum group(Group F)and 3-m A + SQHT containing serum group(group G).After treatment,the cells in each group were detected as follows: the formation of autophagosome was observed by MDC staining;The ultrastructure of 16 HBE cells was observed by TEM;The relative expressions of ATG5,ATG12,beclin-1,Bax,Bcl-2,p62 and LC3-Ⅱ/LC3-Ⅰ were detected by WB and q RT-PCR.Chapter 3: Effect of Shenqi Huatan Formula on regulating autophagy based on m TOR signal pathway in COPD ratsCOPD rat model was established by intratracheal instillation of LPS combined with smoking;After successful modeling,the rats were randomly divided into control group,model group,high-dose Shenqi Huatan Formula group,medium dose Shenqi Huatan Formula group and low-dose Shenqi Huatan Formula group.The high,medium and low dose groups of Shenqi Huatan Formula were given 1.03g/(kg·d)、 2.06g/(kg·d)、5.12g/(kg·d)of Shenqi Huatan Formula by gavage respectively,and the gavage volume was 10ml/kg.The control group and model group were given the same volume of distilled water by gavage for 4 weeks.Observe the general conditions of rats in each group;The pulmonary function of rats was measured;Lung histomorphology was observed by he method;Expression of IL-1 β、IL-6、TNF-α was observed by IHC staining;Serum IL-6 and IL-1β、TNF-α content were detected by ELISA;The relative expressions of ATG5,ATG12,Beclin-1,Bax,Bcl-2,p62,LC3-Ⅱ/LC3-Ⅰ,AKT,PI3 K and m TOR were detected by WB;The relative expressions of beclin-1,p62,Akt,m TOR and PI3 K m RNA were detected by q RT-PCR.ResultsChapter 1: Effect of m TOR gene silencing on autophagy of 16 HBE cells1 m TOR gene silencing inhibits the proliferation and migration of 16 HBE cells and promotes apoptosisThe results of CCK-8 showed that CSE inhibited the activity of 16 HBE cells in a concentration dependent manner.2.5% concentration of CSE and 48 h were selected as the best concentration and time for further study.m TOR si-RNA-2 was selected by q RT-PCR as the best interference sequence for the next experiment.CCK-8 method was used to detect the cell proliferation ability.The results showed that compared with the BC group,the cell proliferation ability of each group decreased(P<0.05,P< 0.01);Compared with the group M,the proliferation ability of si-m TOR group decreased(P<0.05),and the proliferation ability of m TOR activator 3BDO group increased(P<0.05).Transwell test of cell migration showed that compared with BC group,the number of cells penetrating membrane in each group decreased(P<0.05,P<0.01);Compared with group M,the number of membrane piercing cells in si-m TOR group decreased(P<0.05),and the number of membrane piercing cells in 3BDO group increased(P< 0.05).The results of FCM showed that the apoptosis rate of M group and si-NC group was higher than that of BC group(P<0.05,P<0.01);Compared with group M,the apoptosis rate of si-m TOR group was significantly higher(P<0.01),and that of 3bdo group was lower(P<0.05).2 m TOR gene silencing promotes autophagyThe results of MDC staining showed that the cells in BC group and 3BDO group were evenly stained with yellow green fluorescence,and the autophagosomes in M group,sim TOR group and si-NC group were stained with green particles of different sizes and depths,especially in si-m TOR group.The results of IF,WB and q RT-PCR showed that compared with BC group,the expression of PI3 K,AKT,m TOR and p62 decreased(P<0.05,P<0.01),and the expression of LC3Ⅱ/LC3Ⅰ and Beclin-1 increased(P<0.05);Compared with group M,the expression of PI3 K,AKT,m TOR and p62 decreased,and the expression of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 increased in si-m TOR group(P<0.05),the expression of PI3 K,AKT,m TOR and p62 increased,and the expression of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 decreased in 3BDO group(P<0.05).Chapter 2: regulation of Shenqi Huatan Formula on autophagy of 16 HBE cells1 SQHT serum can promote the proliferation of 16 HBE cells within a certain concentration rangeCCK-8 test results showed that SQHT serum promoted the proliferation of 16 HBE cells in a certain concentration range.10% concentration of SQHT medicated serum and 24 h were selected as the best concentration and time for further study.2 SQHT serum inhibits autophagy of 16 HBE cellsMDC staining results showed that 16 HBE cells in the control group(group A)and3-MA+SQHT medicated serum group(group G)were evenly stained with yellow green fluorescence,SQHT medicated serum group(Group C)and 3-MA group(Group E)were evenly stained with yellow green fluorescence,model group(group B)and rapamycin group(Group D),the chromatin of cells was concentrated,the nucleus was broken into dots,and the staining was very uneven,Rapamycin+SQHT containing serum group(Group F)was dyed into green particles with different sizes and dense staining.TEM results showed that autophagy corpuscles were occasionally seen in group A and group G,the number of autophagy corpuscles in group C and group E was less,the number of autophagy corpuscles in group B and group D was significantly increased,and the number of autophagy corpuscles in group F was the second.The results of WB and q RT-PCR showed that compared with group A,the expression levels of ATG5 increased and the expression levels of Bcl-2 and p62 decreased in each group(P<0.05).Except group G,the expression levels of ATG12,Beclin-1,Bax,LC3-Ⅱ/LC3-Ⅰ increased in other groups compared with group A(P<0.05,P<0.01);Compared with group B,the expression levels of ATG5,ATG12,beclin-1,Bax,LC3-Ⅱ/LC3-Ⅰ protein in group D increased and the expression levels of Bcl-2 and p62 decreased(P<0.05).While in other groups,the expression levels of ATG5,ATG12,Beclin-1,Bax,LC3-Ⅱ/LC3-Ⅰ decreased and the expression levels of Bcl-2 and p62increased(P<0.05);Compared with group C,the expression levels of ATG5,ATG12,Beclin-1,Bax,LC3-Ⅱ/LC3-Ⅰ in group D and group F increased and the expression levels of Bcl-2 and p62 decreased(P<0.05),while the expression levels of ATG5,ATG12,Beclin-1,Bax,LC3-Ⅱ/LC3-Ⅰ in group G decreased and the expression levels of Bcl-2 and p62 increased(P<0.05);Compared with group D,the expression levels of ATG5,ATG12,Beclin-1,Bax,LC3-Ⅱ/LC3-Ⅰ protein in Group E,group F and group G decreased,and the expression levels of Bcl-2 and p62 increased significantly(P<0.01);Compared with group E,the expression levels of ATG5,ATG12,Beclin-1,Bax,LC3-Ⅱ/LC3-Ⅰ in group F increased and the expression levels of Bcl-2 and p62 decreased,while the expression levels of ATG5,ATG12,Beclin-1,Bax,LC3-Ⅱ/LC3-Ⅰin group G decreased and the expression levels of Bcl-2 and p62 increased(P<0.05).Chapter 3: Effect of Shenqi Huatan Formula on regulating autophagy based on m TOR signal pathway in COPD rats1 Shenqi Huatan Formula improves lung function in ratsThe results of pulmonary function test showed that the indexes of pulmonary function in each group decreased in varying degrees compared with the control group(P<0.05,P<0.01);Compared with the model group,the FEV in the medium dose group and high dose group was 0.3、FVC、FEV0.3 / FVC index improved(P<0.05);Compared with the low-dose group,the improvement of indexes in the high-dose group was more obvious(P<0.05).2 Shenqi Huatan Formula inhibits the inflammatory level of COPD rats He staining was used to observe the morphology of lung tissue: in the control group,the lung tissue structure was normal,the alveolar structure was complete and the lumen was unobstructed;In the other groups,the small airway was significantly narrowed,the alveolar cavity fused into pieces,the goblet cells increased,and there were a large number of inflammatory cell infiltration around the bronchus and blood vessels.The severity was: model group > low dose group > medium dose group > high dose group.IHC staining showed IL-1 β,IL-6,TNF-α are mainly expressed in the cytoplasm of airway epithelial cells,with clear brownish yellow particles.Compared with the control group,the brownish yellow granules and staining depth in the cytoplasm of lung epithelial cells in each group increased.The severity was: model group>low dose group>medium dose group> high dose group.The results of ELISA showed that compared with the control group,the expression of IL-1β,IL-6,TNF-α in each group was increased(P<0.05,P<0.01);Compared with the model group,after drug intervention,the expression of IL-1 and IL-6 in the low-dose group decreased(P<0.05),TNF-α was no difference in expression(P>0.05),and the expression of inflammatory indexes decreased in medium and high dose groups(P<0.05,P<0.01);Compared with low and medium dose group,the expression of IL-1β,IL-6,TNF-α decreased(P<0.05).3 Shenqi Huatan Formula inhibits autophagy in COPD ratsWB test showed that compared with the control group,the expression of ATG5,ATG12,Beclin-1,Bax and LC3-Ⅱ/LC3-Ⅰ increased,and the expression of Bcl-2,p62,AKT,PI3 K and m TOR decreased(P<0.05,P<0.01);Compared with the model group,the expression of ATG5,ATG12,Beclin-1 and Bax protein decreased,and the expression of Bcl-2,p62,AKT,PI3 K and m TOR protein increased(P<0.05,P<0.01);The expression difference in high dose group was higher than that in medium dose group and lower dose group(P<0.05).q RT-PCR was used to detect the expression of related genes: compared with the control group,the expression of Beclin-1 m RNA increased and the expression of p62,PI3 K,AKT and m TOR m RNA decreased in each group(P<0.05,P<0.01);Compared with the model group,the expression of Beclin-1 m RNA decreased and the expression of p62,PI3 K,AKT and m TOR m RNA increased in each group after drug intervention(P<0.05,P<0.01),and the expression difference in high-dose group was higher than that in medium-dose group > low-dose group(P<0.05).conclusion(1)Shenqi Huatan Formula can be used as an autophagy inhibitor to prevent excessive autophagy of 16 HBE cells stimulated by CSE.(2)Among the low,medium and high dose groups of Shenqi Huatan recipe,the high dose group has the best effect on inhibiting autophagy,reducing the level of inflammation and improving lung function.(3)Shenqi Huatan Formula can up regulate the expression level of PI3K/Akt/m TOR,down regulate the expression level of autophagy markers such as ATG5,ATG12,Beclin-1 and LC3-Ⅱ/LC3-Ⅰ,inhibit the formation of autophagosome and reduce IL-6,IL-1β and TNF-α in COPD rats,the level of inflammation can delay the decline of pulmonary function in COPD rats.