Study On The Mechanism Of Xiaochaihu Decoction In Inhibiting Pulmonary Inflammation During The Recovery Period Of COVID-19

Research background and purposeSince the outbreak of COVID-19 caused by SARS-CoV-2,with the continuous variation of the virus,its pathogenicity has weakened and its infectivity has increased.In addition,with the comprehensive opening of China’s epidemic prevention and control policy,the recovery after infection has become particularly important relative to how to prevent infection.Xiaochaihu decoction,a traditional Chinese medicine classic prescription,has the functions of eliminating pathogenic factors,strengthening the health,and relieving the triple energizer.In the early stage,under the guidance of Professor Bai Changchuan,a famous Chinese traditional medicine in the country,our research group changed ginseng into codonopsis pilosula,and added tangerine peel,astragalus root,schisandra chinensis,and ophiopogon japonicus with the whole prescription to carry out a clinical research.The results proved that Xiaochaihu decoction has a certain improvement effect on the immune function of COVID-19 convalescent patients,but the mechanism is still unclear.Therefore,our study first predicts the target and pathway of Xiaochaihu decoction in the treatment of COVID-19 through the network pharmacology and molecular docking technology;Then,the chemical components of the serum containing Xiaochaihu decoction were identified by liquid chromatography-mass spectrometry,and the material basis of the compound efficacy was analyzed;Finally,the effect of Xiaochaihu decoction containing serum on lung inflammation,differentiation of alveolar epithelial cells and ACE2 protein,the virus target on the surface of alveolar epithelial cells,was explored through cell experiments to verify the inhibition and repair effect of Xiaochaihu decoction on the lung inflammatory injury in the recovery period of COVID-19,as well as the prevention effect on virus infection.Research method(1)Based on the network pharmacology and molecular docking technology,the study on the target and pathway of Xiaochaihu decoction in the treatment of COVID-19 was predicted.TCMSP and Uniprot databases were used to obtain the effective components and potential targets of Xiaochaihu decoction;Use GeneCards and OMIM databases to obtain potential targets of "Corona Virus Disease 2019" disease;The R software VennR script was used to obtain the intersection gene of Xiaochaihu decoction and COVID-19.The STRING database was used to obtain the protein interaction network diagram,and the R software was used to generate the protein target frequency diagram.The network diagram of "drug-component-target" and "drug-target-disease" of Xiaochaihu decoction in treating COVID-19 was obtained by using Cytascape 3.8.2;Apply Network Analyzer to analyze network topology parameters,and screen the active components based on the top five nodes.Apply R software package to obtain GO enrichment and KEGG enrichment.AuTodock4 software was used to dock the top two active ingredients of the compound with the top four target proteins of PPI network and ACE2 protein structure,and PyMol software was used to visualize the results.(2)Study on the identification of the effective components in the serum containing Xiaochaihu decoction by LC-MS.Weigh the medicine of Xiaochaihu decoction,and obtain the Xiaochaihu decoction after boiling and concentration.SD rats were divided into two groups.They were given orally decoction and distilled water respectively in the morning and evening for seven consecutive days.After taking blood from the abdominal aorta,it was placed and centrifuged to obtain the supernatant,and the serum containing Xiaochaihu decoction and blank serum containing medicine were obtained by inactivation and filtration.LC-MS technology was used to detect the effective chemical components of the serum containing Xiaochaihu decoction,and the pharmacological activities of the chemical components were consulted in combination with literature search to analyze the material basis of efficacy.(3)Based on the cell experiment,the effect of serum containing Xiaochaihu decoction on the lung inflammation model was studied.1.To determine the best time and concentration of Xiaochaihu decoction containing serum to interfere with cells.A549 cells were divided into control group and XCH group.First,A549 cells were treated with 5%volume fraction of XCH medicated serum for 12h,24h,48h and 72h respectively,and the cell survival rate was determined by CCK-8.Then the A549 cells were treated with 1%,2.5%,5%,and 10%volume fraction of XCH medicated serum for 48 hours,and the cell survival rate was determined by CCK-8.2.To construct the pulmonary inflammatory cell model.A549 cells were divided into the control group and LPS group.In LPS group,A549 cells were treated with 2.5 mg/L and 5 mg/L LPS for 48 hours.The cell survival rate was determined by CCK-8 and its state was observed by using the light microscope.3.To study the effect and the mechanism of serum containing Xiaochaihu decoction on pulmonary inflammatory cell model.A549 cells were divided into the control group,LPS group and LPS+XCH group.A549 cells were treated with LPS of 5mg/L for 48h in LPS group,and A549 cells were treated with LPS of 5mg/L and XCH containing serum of 5%volume fraction for 48h in LPS+XCH group.Cell survival rate was measured by CCK-8 method,cell state was observed under light microscope,and intracellular inflammatory factor IL-1β and IL-6 mRNA expression were measured by qPCR technology,AKT and NF-κB protein were measured by WB technology,and the nuclear transfer of NF-κB was observed by IF technique.4.To study the effect of serum containing Xiaochaihu decoction on the phenotype transformation of alveolar epithelial cells.A549 cells were divided into the control group and XCH group.In XCH group,A549 cells were treated with 5%volume fraction of XCH medicated serum for 48 hours.The expression of proSP-C and AQP5 protein was detected by IF technique.5.To study the effect of serum containing Xiaochaihu decoction on the expression of ACE2 protein in alveolar epithelial cells.A549 cells were divided into the control group and XCH group.In XCH group,A549 cells were treated with 5%volume fraction of XCH medicated serum for 48 hours.The expression of ACE2 protein was detected by IF technique.Research results(1)Network pharmacology and molecular docking technology predict the multi-target and multi-path analysis of COVID-19 treated by Xiaochaihu decoction.TCMSP and Uniprot database screened 152 effective ingredients of Xiaochaihu decoction and 312 potential targets;GeneCards and OMIM databases screened 2735 potential COVID-19 targets;The R language obtained 132 intersection targets of Xiaochaihu decoctionand COVID-19.The protein target frequency map showed that the core protein targets with higher frequency are AKT1,ALB,TNF,IL-6,TP53,IL-1β,etc,and AKT1 was the most relevant.Network Analyzer analyzed the network topology parameters and screened out the main active ingredients of Xiaochaihu decoction from high to low,which were quercetin,kaempferol,naringin,isorhamnetinβ-Sitosterol,etc.GO enrichment analysis included 126 items in total,and the more concentrated ones were cytokine activity,cytokine receptor binding,DNA-binding transcription factor binding,etc;There were 169 entries of KEGG enrichment.The AGE-RAGE signaling pathway in the complications of diabetes,fluid shear stress and atherosclerosis,IL-17 signaling pathway and TNF signaling pathway were more concentrated.The above related pathways were all related to AKT signaling pathway.Molecular docking showed that AKT1,ALB,TNF had good binding ability with quercetin and kaempferol;Quercetin,kaempferol and ACE2 have good binding ability.(2)Analysis of the effective components in the serum of Xiaochaihu decoction by LC-MS.LC-MS technology identified that the serum containing Xiaochaihu decoction contained 435 chemical components.Among them,the content of chemical components such as zingerone,gingerol,oroxindin,riboflavine,ruscogenin,baicalin,soyacerebroside ii,beta-sitosterol,curcumenol,astramembrannin i,ect may be high.Bupleurum chinense contained 136 kinds of chemical components,pinellia ternate contained 118 kinds,codonopsis pilosula contained 105 kinds,tangerine peel contained 69 kinds,ginger contained 153 kinds,scutellaria baicalensis contained 92 kinds,schisandra chinensis contained 62 kinds,ophiopogon japonicus contained 16 kinds,jujube contained 121 kinds,licorice contained 79 kinds,and many kinds of traditional Chinese medicine contained the same chemical components.(3)Cell experiment was conducted to explore the effect of Xiaochaihu decoction containing serum on pulmonary inflammation model.1.The best induction time of serum containing Xiaochaihu decoction is 48 hours,and the best induction concentration is 5%volume fraction.CCK-8 results showed that when A549 cells were treated with 5%XCH medicated serum for 12h,24h and 72h,there was no significant difference in cell survival rate,but when treated for 48h,the cell survival rate was significantly increased(P<0.01);CCK-8 results showed that 1%,2.5%,5%and 10%volume fraction of XCH medicated serum significantly promoted the survival rate of A549 cells,among which 5%volume fraction of XCH medicated serum group had the highest survival rate(P<0.01).2.A549 cells were induced by LPS at a concentration of 5mg/L for 48h to successfully establish the pulmonary inflammatory cell model.CCK-8 results indicated that 2.5 mg/L LPS had no effect on the survival rate of A549 cells,while the concentration of 5 mg/L had a significant inhibitory effect on the survival rate of A549 cells(P<0.05);The light microscope observation showed that LPS with a concentration of 5mg/L caused toxic damage to the survival state of A549 cells,some cells in the 40-field of vision fell off in pieces.3.The serum containing Xiaochaihu decoction has inhibitory effect on pulmonary inflammatory cell model.CCK-8 results indicated that LPS significantly reduced the number of A549 cells(P<0.01);The observation under the optical microscope shows that in the 40-field and 200-field of vision,LPS caused toxic damage to A549 cells,and some cells fell off in pieces.XCH saved the toxic damage caused by LPS,and the cells adhered to the wall intensively;The results of qPCR showed that LPS significantly increased IL-1β and IL-6 in A549 cells(P<0.01),co-treatment with XCH and LPS inhibited the expression of IL-1β(P<0.05)and IL-6(P<0.01);WB results showed that LPS up-regulated AKT and NF-κB of A549 cells(P<0.05),the co-treatment of XCH and LPS inhibited the increase of AKT(P<0.05)and NF-κB(P<0.01)protein expression induced by LPS;IF results showed that LPS induced the transfer of NF-κB protein from cytoplasm to nucleus,while XCH and LPS co-treatment inhibited the nuclear transfer of NF-κB.4.The serum containing Xiaochaihu decoction has a promoting effect on the transformation of A549 cell phenotype type Ⅱ to type Ⅰ.IF results showed that XCH treatment of A549 cells down-regulated the expression of proSP-C protein(P<0.05)and up-regulated the expression of AQP5 protein(P<0.01).5.Xiaochaihu decoction can inhibit the expression of ACE2 protein in A549 cells.IF results showed that XCH treatment of A549 cells down-regulated the expression of ACE2 protein(P<0.01).Research conclusion(1)Network pharmacology and molecular docking technology predict that the main active ingredients of Xiaochaihu decoction in the treatment of COVID-19 are quercetin and kaempferol,and its mechanism of action mainly includes:1.It may inhibit the expression of inflammatory factors such as IL-1β,TNF-α,IL-6 and IL-8 by acting on AKT signal pathway,thus inhibiting COVID-19 cytokine storm;2.It may inhibit the invasion process of SARS-CoV-2 by highly binding with ACE2 protein.(2)The serum of Xiaochaihu decoction contains 435 chemical components identified by LC-MS:1.The chemical components with higher content may have the effect of inhibiting inflammatory reaction or inhibiting viral infection,and may be the effective components of Xiaochaihu decoction to play a therapeutic role;(2)Ginger,bupleurum,jujube and pinellia ternate contain most chemical components,which may be the main components of traditional Chinese medicine for Xiaochaihu decoction to play a therapeutic role.(3)Cell experiment research shows that Xiaochaihu decoction has a certain effect on the recovery period of novel coronavirus infection,and the mechanisms may be:1.Xiaochaihu decoction medicated serum can effectively inhibit LPS induced pulmonary inflammatory response of A549 cells,inhibit cytokine storm,and inhibit IL-1β And IL-6 expression,its mechanism may be related to the inhibition of AKT/NF-κB signal path;(2)The medicated serum of Xiaochaihu decoction can effectively promote the transformation of A549 cell phenotype type Ⅱ to type Ⅰ,which may promote the repair of alveolar tissue;(3)The serum containing Xiaochaihu decoction has an inhibitory effect on the expression of ACE2 protein in A549 cells,which may play a certain role in preventing the re-infection of the virus.