Study On The Active Ingredients And Mechanisms Of Lianhua Qingwen Formula Against SARS-CoV-2 Invasion

Coronavirus disease 2019(COVID-19)is a serious respiratory infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2).The COVID-19 pandemic has caused significant public health crisis and economic burdens worldwide.SARS-CoV-2 has continued to evolve and mutate,resulting in new and more infectious variants,such as the current Omicron variants,which have demonstrated greater transmission and immune evasion,making it more difficult to prevent and control.Although,vaccines are the most effective means of preventing SARS-CoV-2 infection,the evolution of vaccine-resistant SARS-CoV-2 variants complicated prophylaxis.As for the therapeutic agent to treat COVID-19,there are only a limited number of drugs available for clinical use,such as Molnupiravir and Paxlovid,and the vast majority of drugs exert their therapeutic effects by inhibiting viral replication.Given the growing severity of the epidemic prevention situation and the limited clinical options available for treating COVID-19,the development of alternative drugs for the prevention and treatment of COVID-19 is of paramount importance.Viral inhibitors have the potential to block viral entry and can act as both preventative and therapeutic agents,offering a wider range of clinical applications.As such,the development of broad-spectrum viral entry inhibitors that can prevent and treat COVID-19 is of great practical significance,particularly given the long-term coexistence of the epidemic and the challenges posed by frequent viral mutations.After the outbreak of the COVID-19 pandemic,Lianhua Qingwen capsule(LHQWC)were wildly used as a Chinese patent medicine for treating mild COVID-19 cases and were included in the "Diagnosis and Treatment Plan for COVID-19”.Clinical studies have shown that the use of LHQWC improved the clinical cure rate of patients and prevent close contacts and secondary infections.In addition,in vitro studies have confirmed that LHQWC inhibited the replication of SARS-CoV-2.Previous studies have also demonstrated that LHQWC has broad-spectrum antiviral effects against SARS-CoV,H3N2 influenza virus,respiratory syncytial virus,and other viruses.While LHQWC has shown some effectiveness in preventing and treating COVID-19,a more comprehensive understanding of its pharmacological components and mechanisms for anti-SARS-CoV-2 and broad-spectrum antiviral activity will require further research.Given the highly pathogenic and contagious nature of SARS-CoV-2,experimentation and investigation must be conducted in biosafety level(BSL)3 or 4 laboratories.However,the limited availability of high-level biosafety laboratories in China has hindered research on SARS-CoV-2.Consequently,there is a need to explore SARSCoV-2 substitute models that can be studied in BSL1 or BSL2 laboratories.At the same time,animal or cell models used to simulate SARS-CoV-2 infection differ significantly from the human body and can only simulate some of the characteristics of SARS-CoV2 infection.Thus,it is crutial to develop host models that are closer to the real human condition.To address these challenges,virus-like particles(VLPs)containing all the structural proteins of SARS-CoV-2 have been constructed.These VLPs as replicationincompetent nanoparticles only contain virus structural proteins,making it a noninfectious safe model.As a host model,lung organoids derived from human induced pluripotent stem cells(hiPSC)have the most similar protein expression profile to the real human body and can compensate for the defects of current host models for SARSCoV-2 infection research.PurposeThe study aimed to identify the chemical components of the LHQWC and its medicated serum,investigate the effects of LHQWC on the invasion of SARS-CoV-2 into host cells,determine the specific chemical components derived from LHQWC that can inhibit the invasion of SARS-CoV-2 into host cells,and explain their mechanism of action.Methods1.The LHQWC was administered to SD rats via gavage to obtain its medicated serum.UPLC-MS/MS was used to detect the chemical components of both LHQWC and its medicated serum.The prototype and metabolite components were detected by comparing the components of LHQWC and its medicated serum.To select candidate drugs,we summarized the antiviral effects of the chemical components of LHQWC through literature mining.2.To test the effectiveness of the LHQWC and its chemical components in preventing SARS-CoV-2 infection,SARS-CoV-2 VLPs packaging a luciferase reporter transcripts(Luc-VLPs)were constructed and human ACE2 transgenic C57BL/6J mice were used as the animal model.The formula and its effective components were orally administered by gavage for preventing SARS-CoV-2 infection,and VLPs were administrated through trachea injection to simulate SARS-CoV-2 infection.We assessed the prevention efficacy of LHQWC and its components by measuring the level of SARS-CoV-2 VLPs in the lungs and visualizing its distribution within cells through immunofluorescence staining.Additionally,the effect of the drug on the receptors related to SARS-CoV-2 invasion was determined by detecting the expression levels of ACE2 and TMPRSS2 using immunoblotting.3.To evaluate the effect of the LHQWC and its chemical components on SARS-CoV2 invasion via endocytosis,Vero E6 cells were used as the cell model.The level of SARS-CoV-2 VLPs entry was measured to determine the effectiveness of the drug in inhibiting viral entry.4.Using the dual split protein(DSP)method,a cell model of SARS-CoV-2 mediated membrane fusion was constructed.The LHQWC and its chemical components were used to intervene the membrane fusion process of SARS-CoV-2.The drug’s effect on membrane fusion was determined by measuring the levels of green fluorescent protein and luciferase formed after the cell-cell fusion.Furthermore,the broad-spectrum membrane fusion inhibition effect of the chemical components of LHQWC to SARSCoV-2 variants and other coronaviruses,such as SARS-CoV and MERS-CoV,were also determined by the same method.5.To investigate the effect of kaempferol(Kae)on SARS-CoV-2 entry into alveolar epithelial cells type Ⅱ(AECⅡ),hiPSC were differentiate into AECⅡ.The expression levels of ACE2 and TMPRSS2 in AECⅡ were assessed using immunofluorescence staining.Additionally,AECⅡ was employed to detect the impact of Kae on the invasion of SARS-CoV-2 by enzyme-linked immunosorbent assay(ELISA)and immunofluorescence staining.6.Further research was conducted on the mechanism of action of Kae from LHQWC,which has an inhibitory effect on SARS-CoV-2 membrane fusion.Its targets were identified by intervening at different stages of membrane fusion.Firstly,the level of S-RBD binding to 293F cells expressing ACE2 was detected using flow cytometry,and the effect of Kae intervention on this process was evaluated.Secondly,the study investigated the impact of Kae on TMPRSS2 cleavage activity.HEK-293T cells expressing TMPRSS2 were generated by transient transfection.Kae was administered to the cells during the cleavage process of the fluorescence substrate Boc-QAR-AMC,and the resulting TMPRSS2 enzyme activity was evaluated based on the changes in fluorescence intensity.To simulate the cleavage of the S protein by TMPRSS2,HEK-293F-ACE2 cells expressing TMPRSS2 were mixed with HEK-293T cells expressing SARS-CoV-2 S protein.Kae was introduced during this process,and the generated S2’ fragments after cleavage was detected using immunoblotting to assess the impact of Kae on TMPRSS2 cleavage activity.Molecular docking technology was then used to predict the affinity and binding sites of Kae and TMPRSS2.Surface plasmon resonance(SPR)technology and Cellular Thermal Shift Assay(CETSA)were both used to detect the binding affinity between Kae and different subunits of the S protein.The inhibition effect of Kae on the formation of 6-HB,which is an essential step for membrane fusion,was examined using circular dichroism(CD)to detect changes in the secondary structure of HR1 and HR2.Non-denaturing gel electrophoresis and amino acid mutation technologies were used to assess the impact of Kae on HR1,HR2,and 6-HB formation.Additionally,atomic force microscopy(AFM)was employed to observe the effect of Kae on the stability of HR1,HR2,and 6-HB aggregation under gas-phase conditions.Results1.A total of 129 chemical components from both the raw material and LHQWCmedicated serum were identified.Among them,9 metabolite components were exclusive to the LHQWC-medicated serum.Literature research revealed that 38 of these components possess antiviral properties,including inhibiting viral adhesion,entry,replication,synthesis,release,and direct destruction of viral structure,capable of combating 34 different viruses of Baltimore virus classifications I-VI.2.In vivo study showed that prophylactic administration of LHQWC resulted in a significant reduction of VLPs levels in the lungs,with luciferase signals decreasing to 267 ± 101%compared to the control group(2330 ± 399%,p<0.005).In comparison,the positive control drug,ursodeoxycholic acid(UDCA),reduced VLPs levels to 232± 72%(p<0.005).Immunofluorescence staining revealed a decrease in VLPs particles in lung tissues of LHQWC and UDCA intervention groups.Western blot analysis showed that UDCA intervention lowered ACE2 expression in mouse lungs,while LHQWC did not.These findings suggest that LHQWC effectively prevents SARSCoV-2 infection without altering host ACE2 expression level.3.LHQWC and its medicated serum were found to inhibit both the endocytosis and membrane fusion of SARS-CoV-2.From the 129 chemical components in LHQWC,17 compounds were selected for further study based on their reported antiviral effects.These compounds include forsythoside I,forsythoside A,rhein,glycyrrhizic acid,neochlorogenic acid,phillyrin,salidroside,pogostone,Kae,rutin,diosmin,diosmetin,aloe-emodin,prunasin,dryocrassin ABBA,luteolin and a-linolenic acid.However,only Kae demonstrated a significant inhibitory effect on the membrane fusion process mediated by SARS-CoV-2 S,ACE2,and TMPRSS2,while the other 16 compounds did not inhibit the viral entry process.4.In vivo experiments showed that Kae effectively decreased the level of VLPs in the lungs to 1944± 103%compared to the control group(2480± 220%,p<0.005),indicating its potential to prevent SARS-CoV-2 infection.Further experiments involving immunofluorescence staining and protein immunoblotting showed a significant reduction in the number of VLP particles in the lung tissue of the Kae treatment group,while the expression level of ACE2 in mice lung remained unaffected.In vitro cell experiments using AECⅡ also confirmed that Kae reduced the level of VLPs by 46.3±15.8%(p<0.005)and exhibited inhibitory effects on the invasion of SARS-CoV-2 particles into AECⅡ..5.Kae demonstrated inhibitory effects on SARS-CoV-2 invasion primarily through its suppression of viral fusion rather than endocytosis.It significantly inhibited the membrane fusion process mediated by S protein,ACE2,and TMPRSS2(p<0.005),as well as the membrane fusion of SARS-CoV-2 variants,including Delta,Omicron variants(BA.1,BA.2,BQ.1.1,XBB.1),SARS-CoV,and MERS-CoV.The mechanism studies revealed that Kae did not impact the binding between SARS-CoV-2 S-RBD and ACE2,but it did impede the activity of TMPRSS2(p<0.005).Kae interacted with the S protein,specifically the S2 subunit,and hindered the formation of 6-HB by degrading HR1 and HR2.Additionally,Kae disrupted the α-helical conformation of HR1,as detected by CD,and destabilized the structural integrity of HR1 and HR2,as observed through non-denaturing gel electrophoresis detection and AFM imaging.The analysis of amino acid mutations suggested that Kae’s action targeted the lysine residue in HRs.Conclusions1.The use of replication-deficient SARS-CoV-2 Luc-VLPs together with human ACE2 transgenic mouse animal models and AECⅡ models can partially reproduce the biological process of SARS-CoV-2 viral particle invasion into host cells,which can be used to study the effects of drugs on viral invasion.2.LHQWC and its chemical component Kae both have the efficacy of preventing viral infections by inhibiting the invasion of SARS-CoV-2.LHQWC exhibits inhibitory effects on both viral endocytosis and membrane fusion process,while Kae mainly inhibits membrane fusion to achieve the effect of blocking virus invasion.3.Kae targets the lysine residues in the HR region of the S2 subunit to disrupt the HR structure stability,thereby inhibiting the formation of 6-HB and ultimately suppressing membrane fusion.The presence of lysine residues has been shown to significantly affect the stability of the HR structure,which has important implications for the development of effective broad-spectrum membrane fusion inhibitors.