CN113197981A

CN113197981A - Application of Shuqing granules in preparation of medicine for treating novel coronavirus pneumonia

Info

Publication number: CN113197981A
Application number: CN202110628320.1A
Authority: CN
Inventors: 王放; 王国强; 国晓娉; 李生; 张春梅; 杜鹤; 刘传贵
Original assignee: Jilin Huakang Pharmaceutical Co ltd
Current assignee: Jilin Huakang Pharmaceutical Co ltd
Priority date: 2021-06-03
Filing date: 2021-06-03
Publication date: 2021-08-03
Anticipated expiration: 2041-06-03
Also published as: CN113197981B

Abstract

The invention relates to application of Shuqing granules in preparation of a novel medicine for treating coronavirus pneumonia, and belongs to the field of medicines. The application of the sparse clear particle in preparing the medicine for treating the novel coronavirus pneumonia can obviously reduce the IL-6 secretion of Calu-3 cells and the phosphorylation level of NF-kappa Bp65 protein caused by the stimulation of SARS-CoV-2S protein, and the sparse clear particle can regulate NF-kappa B signal passage by competitively binding with receptors of SARS-CoV-2 on cells and play a role in resisting inflammatory reaction induced by SARS-CoV-2. The method has the advantages that the network pharmacology and the molecular docking combined cell experiment method are adopted, the anti-SARS-CoV-2 action mechanism of the Shuqing particles is preliminarily predicted and verified, the experimental basis is provided for the Shuqing particles to exert the anti-SARS-CoV-2 activity, and the theoretical basis is provided for the subsequent research.

Description

Application of Shuqing granules in preparation of medicine for treating novel coronavirus pneumonia

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of Shuqing granules in preparation of a novel medicine for treating coronavirus pneumonia.

Background

The Chinese medicine is a treasure of Chinese nation, has been developed and advanced for thousands of years, and plays an extremely important role in preventing and treating SARS, various viral influenza, Ebola and other virus infectious diseases. At present, scholars at home and abroad report about more than 200 Chinese medicaments with antiviral activity, but the pharmacological mechanism of the Chinese medicaments is not clear, and the development of the Chinese medicaments is seriously inhibited. Therefore, screening the antiviral effective components of traditional Chinese medicine and analyzing the molecular pharmacological mechanism thereof have become one of the hot spots for developing new antiviral drugs.

The Shuqing granules are produced by Jilin Huakang pharmaceutical industry GmbH, approved document No: the Chinese medicine Z10980132 is a Chinese patent medicine containing folium isatidis, folium mori, rhizoma phragmitis, liquorice, gypsum and other components, and has the effects of ventilating lung and stomach, clearing heat and detoxicating. The study showed that: the dyers woad leaf is dry leaf of Chinese traditional Chinese medicine isatis indigotica fort, is bitter in taste and cold in nature, has the effects of cooling blood, relieving sore throat, clearing heat and removing toxicity, and is mainly used for treating fever, headache or macula, carbuncle swelling, sore and sore, sore throat and the like. Clinical researches find that the Chinese medicinal composition has obvious treatment effect on viral diseases such as influenza, acute infectious hepatitis, epidemic encephalitis B, epidemic parotitis, herpes simplex viral keratitis and the like. Other researches indicate that the compound can obviously inhibit virus proliferation and improve the immunity of the organism. The mulberry leaves have the effects of dispelling wind and heat, clearing lung-heat, moistening dryness, clearing liver and improving eyesight, and are listed as one of the ten health-care foods in the 21 st century for human beings by the international food health organization at present. The 1-deoxynojirimycin in the mulberry leaves has broad-spectrum high-efficiency antiviral activity, can obviously inhibit the activity of retrovirus and has obvious antiviral effect. In vitro experiments prove that mulberry leaves have direct inactivation effect on respiratory syncytial virus. The reed rhizome is one of the clinical common traditional Chinese medicines and has the functions of relieving fever and pain, clearing heat and promoting fluid production. The licorice has the effects of tonifying middle-jiao and Qi, relieving spasm and pain, clearing away heat and toxic materials, eliminating phlegm and stopping cough and harmonizing drug property. The liquorice is commonly used in the traditional Chinese medicine for relieving cough and eliminating phlegm, and treating respiratory diseases such as cough, bronchitis, pneumonia and the like. There is over 200 years history in western countries for treating excessive phlegm cough and asthma with radix Glycyrrhizae extract and compound radix Glycyrrhizae mixture. And the traditional Chinese medicines recommended in the novel diagnosis and treatment scheme for coronavirus pneumonia, such as Lianhua Qingwen Capsule, Maxingshigan decoction, Agastache Capsule, Lung heat-clearing and toxin-expelling decoction, wind-dispelling and toxin-expelling Capsule, Dayuan drink, Qing Wen Baidu drink, Tankejing, etc., all contain licorice.

The concept of cyber pharmacology was first introduced in 2007 by Andrew l.hopkins, a british scholars, Nature Biotechnology, and aims to explain the disease process by establishing a database, constructing a network, analyzing the network and then performing experimental verification. The novel cross discipline is a new cross discipline which takes multidirectional pharmacology and system biology as theoretical bases, integrates multi-discipline ideas such as bioinformatics, network topology analysis and the like, and carries out treatment target screening and analysis and new drug design. Network pharmacology has become an important theoretical tool for assisting in discussing complex action mechanisms of Chinese and western medicines in recent years, and system mapping is carried out on target interaction behind the treatment effect of global statistics, so that system-level understanding of potential candidate medicine molecular targets is provided, the influence mode and intervention approach of medicines on diseases are clarified, and the mechanism of synergistic action of medicines on the diseases is analyzed from a new perspective, which is a necessary premise of a phenotype-based medicine discovery process.

The computer virtual screening technology is an important method for computer-aided drug design and high-throughput screening of new drugs, and is essentially a process for searching new ligands based on biological structures, so that the process greatly reduces the research range of ligand screening by adopting an artificial method. At present, as an important auxiliary means in the field of new drug research, a computer virtual screening technology based on molecular docking plays an extremely important role in researching the action mechanism between drugs and targets and developing new drugs.

The molecular docking technology is essentially to dock some small molecules at active binding sites of biological macromolecules, can find the three-dimensional structures of the small molecules in some known structure databases, then optimizes receptor molecular conformation, ligand receptor connection positions and the like through a series of crystal structure processing processes of crystal water removal, missing hydrogen atom supplement, missing information repair and the like, finally finds the optimal conformation for combining the ligand small molecules and the target macromolecules, further scores the docked result, selects the optimal ligand in a plurality of small molecules according to the scored result, and then further applies the optimal ligand to an entity drug screening experiment.

At present, molecular docking methods are mainly divided into three types including rigid docking, semi-flexible docking and flexible docking. The rigid docking means that the spatial conformation of a receptor and a ligand participating in docking is not changed in the docking process, and the position and the posture of the receptor and the ligand in the space are only adjusted and then docking is carried out. When docking is carried out by this method, since the spatial conformation of the receptor and the ligand is regarded as stable, the docking speed is relatively high when docking is carried out by this method, and thus docking between macromolecules such as proteins and nucleic acids is suitably carried out. Secondly, semi-flexible butt joint: when docking is performed, the receptor conformation is a rigid conformation, while the ligand conformation can be subjected to a series of adjustments within a certain range. Therefore, the method is more suitable for processing the docking between the small molecular ligand and the large molecular receptor, is a common docking method in the current molecular docking process, and is widely applied to the aspects of new drug development, design and the like. Flexible butt joint: during docking, the spatial conformation of the ligand and receptor can be altered as desired. However, the flexible docking method has relatively high requirements on a software system, and has very accurate requirements on three-dimensional space conformations of ligand and receptor molecules, so that the calculation amount is extremely large, and the efficiency is low. Because the docking result can be calculated most accurately, the method is more suitable for accurately researching the recognition condition between the ligand molecule and the receptor molecule.

Disclosure of Invention

The invention provides application of Shuqing granules in preparing a medicament for treating novel coronavirus pneumonia.

The invention adopts the technical scheme that: an application of Shuqing granule in preparing medicine for treating coronavirus pneumonia is disclosed.

The dredging particle can obviously reduce the IL-6 secretion of Calu-3 cells and the phosphorylation level of NF-kappa Bp65 protein caused by the stimulation of SARS-CoV-2S protein.

The Shuqing particle provided by the invention regulates NF-kB signal channels by competitively binding receptors of SARS-CoV-2 on cells, and plays a role in resisting inflammatory response induced by SARS-CoV-2.

When the invention is used for preparing the medicine for treating the novel coronavirus pneumonia, the oral administration or the non-oral administration is safe. In case of oral administration, it may be administered in any conventional form, such as powder, granule, tablet, capsule, pill, drop pill, soft capsule, floating agent, oral liquid, suspension, syrup, buccal tablet, spray or aerosol, etc. when the drug is not administered orally, any conventional form may be employed, such as suppository, injection intravenous injection, intramuscular injection, ointment, inhalant, etc.

The present invention is constituted by a solid or liquid excipient, which is well known in the art, and examples of the solid excipient include lactose, starch, dextrin, calcium carbonate, synthetic or natural aluminum sulfate, magnesium chloride, magnesium stearate, sodium bicarbonate, dried yeast, and other liquid excipients such as water, glycerin, propylene glycol, simple syrup, ethanol, ethylene glycol, polyethylene glycol, sorbitol, and other ointments, and fatty oils, and hydrophobic or hydrophilic agents composed of aqueous lanolin, vaseline, glycerin, beeswax, wood wax, liquid paraffin, resin, and higher waxes.

The invention has the advantages that the network pharmacology and the molecular docking combined cell experiment method are adopted, the anti-SARS-CoV-2 action mechanism of the Shuqing particle is preliminarily predicted and verified, the experimental basis is provided for the Shuqing particle to exert the anti-SARS-CoV-2 activity, and the theoretical basis is provided for the subsequent research.

Drawings

FIG. 1 shows the expression of ACE2 protein in different cells;

figure 2 is the effect of different concentrations of clearance particles on Calu-3 cell activity, P <0.001, P < 0.0001;

FIG. 3 is the change in the amount of IL-6 secretion from Calu-3 cell supernatant after protein stimulation, P < 0.05;

FIG. 4 is a graph of the change in NF-. kappa.Bp 65 protein and its phosphorylation levels in Calu-3 cells following stimulation with SARS-CoV-2S protein,. times.P < 0.05;

FIG. 5 is a graph of the effect of clearing particles on the change in IL-6 secretion from Calu-3 cells following stimulation with SARS-CoV-2S protein,. P < 0.05;

FIG. 6 is a graph showing the effect of Westernblot method on the phosphorylation level of NF-. kappa.Bp 65 protein in Calu-3 cells after stimulation with SARS-CoV-2S protein by using different concentrations of borrelidin, wherein A, WesternBlot is a bar graph; B. relative gray values for each set of bands were calculated from graph a,. P < 0.05.

Detailed Description

The invention adopts network pharmacology and molecular docking combined in vitro cell experiments to develop pharmacodynamic research of anti-SARS-CoV-2 of the Shuqing granules, and obtains 12 key genes by constructing active components of the Shuqing granules, namely COVID-19 target spot network screening: VEGFA, POR, PLAT, IL6, IL10, HMOX1, GPT, ENPEP, EGFR, CTSB, CRP, ACE, and further screening according to topological parameters such as degree values to obtain 13 key active ingredients, which is shown in Table 1.

TABLE 1 Key active ingredient information Table

Obtaining a possible pathway of dredging particle therapy COVID-19 through GO analysis and KEGG analysis, and analyzing a key pathway by combining literature; according to the results of network pharmacology screening analysis, key genes and key active ingredients are subjected to batch butt joint, and the results show that the four active ingredients of indirubin, kaempferol, 4, 5, 7-trihydroxyflavanone and (R) -2-hydroxypropionic acid have strong hydrogen bond effects with four receptor proteins of ACE2, CRP, EGFR and IL 10. The results indicate that the hydrophobic particles may bind to multiple receptors through strong hydrogen bonding to exert an anti-COVID-19 effect.

The effects of the present invention will be further described below by way of experimental examples.

Experimental example 1 cell Effect of Shuqing particles on SARS-CoV-2S protein stimulation

1.1 Experimental materials

1.1.1 Experimental cells and Shuqing granules

(1) Calu-3 cells: calu-3 is a human lung adenocarcinoma cell cryopreserved by the basic medical college of Jilin university.

(2) A549 cells: a549 cells were adenocarcinoma human alveolar basal epithelial cells cryopreserved by the basic medical college of the university of gilin.

(3)16HBE cells: is human bronchial epithelial-like cells, cryopreserved by the basic medical college of Jilin university.

(4) BEAS-2B cells: is a normal lung epithelial cell of human, and is cryopreserved by the basic medical college of Jilin university.

(5)293T cells: the derivative of human embryonic kidney cell line 293 inserted with SV 40T-antigen gene and having high transfection efficiency is called 293T, and is cryopreserved by the basic medical college of Jilin university.

(6) Dredging particles: jilin Huakang pharmaceutical industry, Inc., approved document number: the national standard character number Z10980132.

(7) SARS-CoV 2S protein: the SARS-CoV-2 genome encodes one of four major structural proteins, available from Beijing Yinqiao (Sino Biological Inc)

1.1.2 Experimental reagents

TABLE 2 major reagents

1.1.3 Experimental instruments

TABLE 3 Main instruments

1.2 Experimental methods

1.2.1 cell culture

(1) Cell recovery: preheating a water bath kettle to 37 ℃ in advance, taking out the cells cryopreserved in a liquid nitrogen tank, quickly placing the cells in the water bath kettle at 37 ℃, continuously shaking the cryopreserving tube, centrifuging (1000rpm for 5min) when few parts in the tube are crystallized, transferring the cell cryopreserved in an ultraclean workbench for aseptic operation, discarding supernatant, adding 1mL of culture solution into the precipitate, gently and repeatedly blowing and uniformly mixing the cell cryopreserving tube to form cell-free blocks, transferring the cell cryopreserving tube into an aseptic centrifuge tube, adding 5mL of culture solution into the centrifuge tube for continuous uniform mixing, transferring the cell cryopreserving tube into a disposable cell culture dish, placing the cell culture dish into a cell culture box (37 ℃, 5% CO and 5 ℃ C.), and placing the cell culture box into a cell culture box₂) And (5) culturing.

(2) Cell passage: cell passage is carried out when the cell growth density in the culture dish is observed to be about 80% -90%. The original culture medium is firstly sucked and discarded by a pipette, after being washed for 3 times by sterile PBS, 2mL of 0.25% pancreatin solution is added and placed in an incubator for digestion, and when most cells are observed to shrink and become round under a microscope, 1mL of culture solution is added to stop digestion. The cells attached to the dish were gently pipetted down and transferred in their entirety to a sterile centrifuge tube for centrifugation (1000rpm, 5 min). Abandoning the supernatant, resuspending the cells with cell culture fluid, and subpackaging to 2-3 new cell culture dishes for continuous culture as required.

(3) Freezing and storing cells: when the cells are in the logarithmic growth phase and have good shapes, firstly, a pipettor is used for sucking and removing the original culture medium, after the cells are washed for 3 times by sterile PBS, 2mL of 0.25% pancreatin solution is added and placed in an incubator for digestion, and when most of the cells are observed to shrink and become round, 1mL of culture solution is added to stop digestion. The cells attached to the dish were gently pipetted down and transferred in their entirety to a sterile centrifuge tube for centrifugation (1000rpm, 5 min). Discarding the supernatant, adding appropriate amount of cell freezing medium (DMSO: cell culture medium: serum 7: 2: 1), subpackaging into freezing tubes, placing into cell freezing box, freezing at-80 deg.C for 24 hr, and transferring into liquid nitrogen tank for storage.

1.2.2 Total protein extraction and protein concentration determination

(1) Cell protein extraction

A. Preparation of cell lysate: each 1mL of lysate contained 10. mu.L of LPMSF, 10. mu.L of phosphatase inhibitor, and 980. mu.L of LRIPA.

B. The original cell culture medium was aspirated and washed 3 times with pre-cooled PBS buffer.

C. The six well plate was placed on ice, 150 μ L of protein lysate was added to each well, and the lysate was blown up with a pipette to make full contact with the cells.

D. Transferring the liquid into EP tube, placing on ice, vortexing once every 10min, repeating for three times, centrifuging (4 deg.C, 12000g, 5min), and collecting supernatant as total cell protein.

(2) Protein concentration determination

Preparing a BCA working solution: and (3) mixing the solution A and the solution B in the BCA kit according to the ratio of 50: 1, mixing the above materials, and mixing the above materials.

B. Preparation of protein standard solution: the protein standards were completely dissolved in PBS and diluted to concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL, respectively.

C. Loading: and adding 200 mu LBCA working solution, 20 mu L of protein standard substance with each concentration and a sample to be detected into each hole of a 96-hole plate, and uniformly mixing.

D. And (3) incubation: the 96-well plate was incubated at 37 ℃ for 30 min.

E. And detecting the OD value at the 562nm wavelength by using a microplate reader, drawing a standard curve, and calculating the protein concentration of the sample to be detected.

1.2.3 protein immunoblotting (Westernblot)

(1) Adding 4mL of separation glue into the fixed clean rubber plate, then quickly injecting absolute ethyl alcohol above the fixed clean rubber plate to ensure that the rubber block is horizontally bubble-free, after the separation glue is solidified, obliquely pouring out the absolute ethyl alcohol, adding the concentrated glue, slightly inserting the glue comb, and waiting for the concentrated glue to be solidified.

(2) Protein sample pretreatment: adding 5 Xloadingbuffer into the sample according to the proportion, mixing uniformly, putting into boiling water for 10min, and storing at 4 ℃ until the room temperature is recovered for later use.

(3) Loading: and (3) installing an electrophoresis tank and a rubber plate to ensure no leakage, adding electrophoresis liquid into the electrophoresis tank until the liquid level overflows the outer glass plate, slightly pulling off a rubber comb, slightly blowing a sample hole by using a pipettor, slowly adding a processed protein sample, and adding a protein Marker into the holes on two sides.

(4) Electrophoresis: and (3) switching on a power supply, carrying out electrophoresis at a constant voltage of 80V until the sample is compressed to the same position and reaches the junction of the separation gel and the concentrated gel, raising the voltage to 120V, and continuing the electrophoresis until the bromophenol blue indicator reaches the lower edge of the separation gel.

(5) Film transfer: after electrophoresis, the sponge pad and the filter paper are soaked in a membrane transferring solution in advance, a strip is cut according to the indication position of a protein Marker strip, the PVDF membrane is cut according to the size of the gel, and the PVDF membrane is soaked in methanol for 30s to be activated. Then, a membrane transferring splint is correctly assembled according to the sequence of the negative electrode, the spongy cushion, the filter paper, the gel, the PVDF membrane, the filter paper, the spongy cushion and the positive electrode, the membrane transferring splint is placed into a membrane transferring groove, membrane transferring liquid is fully added, and the ice bath is performed at constant current for 100mA 1 h.

(6) And (3) sealing: the PVDF membrane was taken out, washed with TBST for 5min, repeated three times, and then blocked by adding 5% BSA solution at room temperature for two hours.

(7) Primary antibody incubation: the primary antibody was diluted as indicated with 5% BSA solution to the appropriate concentration. The blocking solution was discarded, and the blocked PVDF membrane was placed in the corresponding primary antibody and incubated overnight at 4 ℃.

(8) Washing the membrane: the first antibody was recovered the next day and stored at 4 ℃. PVDF membrane was washed 6 times with TBST buffer for 5min each.

(9) And (3) secondary antibody incubation: and (3) diluting the secondary antibody to an appropriate concentration by using TBST, putting the cleaned PVDF membrane into a corresponding secondary antibody solution, and incubating for 1h at room temperature.

(10) Washing the membrane: the secondary antibody was aspirated and the PVDF membrane was washed 6 times 5min each with TBST buffer.

(11) And (3) developing: mixing ECL developing solution A, B according to the ratio of 1:1, and preparing the mixture on site. And soaking the PVDF film in a developing solution, slightly shaking to ensure that the film is fully contacted with the liquid, and then exposing and developing the PVDF film by using a gel imager.

(12) Gray value analysis: and calculating the gray value of each strip by using Image J Version6.0 software for subsequent analysis.

1.2.4 CCK8 method for determining maximum nontoxic concentration of cells

Weighing 5g of Shuqing granules, dissolving in 100mL of cell maintenance solution preheated at 37 ℃, filtering once by using a sterile filter head with the diameter of 0.45 mu m, and then filtering once by using a sterile filter head with the diameter of 0.22 mu m to obtain 50mg/mL of Shuqing granule liquid as mother liquor. Respectively diluting the mother liquor according to concentration gradient to 8 liquid medicines with concentration of 40mg/mL, 30mg/mL, 20mg/mL, 10mg/mL, 5mg/mL, 1mg/mL and 0.1mg/mL of the diluted solution and the mother liquor for standby application, and preparing the liquid medicines for use. Taking the cell suspension in logarithmic growth phase, diluting to 5X 10⁴-1×10⁵Per/mL, 100. mu.L of diluted cell suspension was added to each well of a 96-well plate, and the mixture was incubated at 37 ℃ with 5% CO₂The inside of the cell culture box is 24 hours. Discarding supernatant, adding 100 μ L of each concentration of medicinal liquid according to concentration gradient, and adding 100 μ L of maintenance liquid into the wells with blank control group. After 24h of drug action, the medium was discarded and 100. mu.L of the maintenance medium and 10. mu.L of LCCK8 reagent were added to each well. Then placing the mixture into an incubator to incubate for 1-4h, and measuring OD₄₅₀Calculating the maximum nontoxic concentration (TC) of the drug₀) This was repeated three times.

1.2.5 real-time unlabeled cell function Analyzer (RTCA) for detecting cell growth index

Cell growth indices were recorded using RTCA software based on the live cell resistance values. The cell suspension was diluted to 1X 10 as specified⁴Per mL, 100. mu.L of cell suspension per well was added to the plate. After 24h, the culture solution is aspirated, and the diluted sparse and clear granule liquid medicine is added according to the concentration gradient, wherein each hole is 100 mu L. And after 48h, observing the cell growth index of each hole, calculating the relative activity of the cells, and selecting the maximum non-toxic concentration to perform subsequent experiments by combining the CCK8 experiment results.

1.2.6 enzyme-linked immunosorbent assay (ELISA)

(1) Taking the cell suspension in the logarithmic growth phase, inoculating the cell suspension in a 6-well plate, and culturing until the cell growth density is about 80-90%.

(2) Cells were carefully washed 2-3 times with PBS, different concentrations of drug were added, and a blank control was set.

(3) The cell supernatant was collected, centrifuged (4 ℃, 12000g, 15min), and the supernatant was collected.

(4) The ELISA detection kit was equilibrated at room temperature for 30 min.

(5) And adding the standard substance diluent into the standard substance tube, and uniformly mixing by vortex to prepare a mother solution. Diluting the mother solution by times according to the kit instruction to prepare the standard substance with different concentration gradients.

(6) And respectively adding a gradient concentration standard substance and a sample to be detected into the antibody-coated microporous plate, wherein each hole is 100 mu L.

(7) Adding 50 μ L biotin-binding antibody into each well, sealing, slowly shaking and mixing on a shaking table, and incubating at room temperature for 2-3 h.

(8) The supernatant was discarded and washed three times with 300. mu.L 1 Xwash per well, and after the third wash was blotted dry on filter paper.

(9) Add 100. mu.L of horseradish peroxidase (HRP) -labeled detection antibody to each well, seal the plates, and incubate on a shaker for 1h at room temperature.

(10) And (4) discarding the supernatant, and repeating the step (8) for cleaning.

(11) Add 100. mu. LTMB reaction substrate per well, seal plate, incubate 30min at room temperature in the dark.

(12) Adding 50 mu L of stop solution into each hole, fully and uniformly mixing, detecting the OD value at the position with the wavelength of 450nm by using an enzyme-labeling instrument, drawing a standard curve, and calculating the content of the corresponding cell factor in the sample to be detected.

1.2.7 data processing

All experimental data in this study were analyzed using Graphpad prism v6.0. The data were first tested for homogeneity of variance and normal distribution and then analyzed using the t-test. P <0.05 showed statistical differences, P <0.01 showed statistically significant differences. Wherein, the Westernblot bands were subjected to gray value analysis using Image J Version 6.0.

1.3 results of the experiment

1.3.1 cellular Screen with high expression of ACE2 protein

The molecular docking result shows that ACE2 is one of the active component binding receptors of the Shuqing granules and is also a SARS-CoV-2S protein binding receptor, therefore, in order to research whether the Shuqing granules can inhibit the stimulation effect of SARS-CoV-2S protein on cells, firstly, cell strains of the high expression S protein binding receptor ACE2 are screened, A549, 16HBE, BEAS-2B, Calu-3 and 293T cells are respectively inoculated in a cell culture dish, when the cell growth density in the culture dish is observed to be about 80-90%, the total protein of various cells is respectively extracted, and the ACE2 expression condition in various cells is detected by a Westernblot method, and the result is shown in figure 1, and the ACE2 expression amount of the Calu-3 cells is higher compared with other four cells. Therefore, Calu-3 cells were used for subsequent experiments in this study.

1.3.2 toxicity of Shuqing granules to Calu-3 cells

In order to determine the optimal administration concentration of the borrelidin on the effect of Calu cells, the toxicity of the borrelidin on the cells is determined by the CCK8 method, and the results are shown in figure 2, wherein the cell survival rate is remarkably reduced (P < 0.01) when the drug concentration is more than 30 mg/mL; when the concentration is less than 20mg/mL, there is no difference in cell viability as compared with the control group. Further analysis shows that the maximum nontoxic concentration of the medicine on cells is TC₀＝20。

The subsequent experiments were carried out with low, medium and high concentrations selected from 5mg/mL, 10mg/mL and 20 mg/mL.

1.3.3 optimal concentration and time screening of SARS-CoV-2S protein stimulated Calu-3 cells

IL-6 is one of the 12 clear granule active ingredient-COVID-19 intersection targets confirmed by the previous research, is also one of the important inflammatory cytokines induced by SARS-CoV-2 induced COVID-19 pneumonia, and is mainly induced by NF-kB classical pathway activation. Therefore, in order to determine the optimal conditions for stimulating Calu-3 cells by SARS-CoV-2S protein, Calu-3 cells were stimulated for 6h and 24h with S protein at concentrations of 100, 200, 400, 800, and 1000ng/mL, respectively, IL-6 levels in cell supernatants were measured by ELISA, and NF-. kappa.Bp 65 protein phosphorylation levels were measured by WesternBlot for comprehensive assessment.

(1) Change of IL-6 secretion amount of Calu-3 cell after SARS-CoV-2S protein stimulation

As shown in FIG. 3, the results show that the amount of IL-6 secretion increases with the increase in the concentration of S protein. Compared with the S protein stimulated 6h group, the IL-6 secretion amount of the stimulated 24h group is integrally increased. IL-6 secretion was significantly increased in the 1000ng/mLS proteome at both time points compared to the blank (P <0.05, P < 0.01). The IL-6 secretion amount of the 24h group stimulated by the 1000ng/mLS protein is also obviously increased (P is less than 0.05) compared with that of the 6h group stimulated by the S protein at the same concentration, so that the 24h stimulated by the 1000ng/mLS protein is the optimal stimulation condition.

(2) NF-kappa Bp65 protein in Calu-3 cells after SARS-CoV-2S protein stimulation and change of phosphorylation level thereof

As shown in FIG. 4, the phosphorylation levels of NF- κ Bp65 protein were increased in each group of cells after S protein stimulation compared to the control group. The increase of the phosphorylation level of NF-kappa Bp65 protein is most obvious when the concentration of S protein is 1000ng and the stimulation time is 24h compared with the control group (P < 0.01).

In conclusion, the experiment selects the Calu-3 in vitro research cell model with the concentration of the S protein of 1000ng and the stimulation time of 24h for constructing the SARS-CoV-2S protein stimulation.

1.3.4 Effect of Shuqing granules on IL-6 secretion from Calu-3 cells after SARS-CoV-2S protein stimulation

In order to know whether the Shuqing granules can inhibit the stimulation effect of SARS-CoV-2S protein on Calu-3 cells, the Shuqing granules with different concentrations are respectively added into the determined cell models, meanwhile, a positive control group of the lotus antipyretic (LH) is set, and the IL-6 level in the cell supernatant of each group is detected. As a result, as shown in FIG. 5, the secretion amounts of IL-6 were increased in the S protein model group, the S protein-low concentration administration group, the S protein-medium concentration administration group and the S protein-high concentration administration group, respectively, as compared with the blank control group. Compared with the S protein model group, the IL-6 secretion of the S protein-high concentration administration group and the S protein-lotus antipyretic administration group are both reduced remarkably (P is less than 0.05). Therefore, the Shuqing granules can obviously inhibit the secretion of IL-6 after S protein stimulation.

1.3.5 Effect of Shuqing granules on the NF-kappa Bp65 protein of Calu-3 cells and its phosphorylation level after SARS-CoV-2S protein stimulation

A large number of documents report that the expression of IL-6 is closely related to the activation of an NF-kappa B signal channel, and the transcription of intracellular target genes is caused by the phosphorylation of NF-kappa Bp65 in an NF-kappa B classical activation path, so that the expression levels of NF-kappa Bp65 and phosphorylated proteins in various groups of cells are detected by adopting WesternBlot in order to discuss whether the effect of borrelidin on the generation of IL-6 is related to the phosphorylation change of NF-kappa Bp 65. As shown in FIG. 6, compared with the S protein model group, the phosphorylation levels of NF-. kappa.Bp 65 protein were not significantly changed in the S protein-low concentration and S protein-medium concentration administration groups, whereas the phosphorylation levels of NF-. kappa.Bp 65 protein were significantly decreased in the S protein-high concentration administration group and the S protein-lotus antipyretic administration group (P < 0.05). Therefore, the borrelidin particles can obviously inhibit the phosphorylation level of NF-kappa Bp65 protein after S protein stimulation, thereby possibly influencing the secretory production of IL-6.

1.4 summary

(1) ACE2 was highly expressed in human lung adenocarcinoma cells Calu-3.

(2) The Shuqing granules can obviously reduce the IL-6 secretion of Calu-3 cells and the phosphorylation level of NF-kappa Bp65 protein caused by the stimulation of SARS-CoV-2S protein.

Experiments show that the Shuqing granules can be possibly combined with ACE2 through competition with S protein of SARS-CoV-2, thereby inhibiting the stimulation effect of the S protein on Calu-3 cells, and reducing IL-6 secretion to play a role in resisting inflammatory reaction caused by SARS-CoV-2 by down-regulating NF-kB signal path.

Based on the above results, the present experiment further develops the pharmacodynamic mechanism of the cell experiment from the biological perspective to verify the anti-inflammatory response of the Shuqing granules to SARS-CoV-2. The experimental result shows that the compound traditional Chinese medicine Shuqing granules can obviously reduce the IL-6 secretion amount caused by S protein stimulation and the phosphorylation level change of NF-kappa Bp65 protein, and the compound traditional Chinese medicine Shuqing granules are suggested to play a role in resisting SARS-CoV-2 induced inflammatory reaction by competitively binding SARS-CoV-2 receptor on cells and regulating NF-kappa B signal path.

Claims

1. Application of Shuqing granule in preparing medicine for treating coronavirus pneumonia is provided.

2. Use of the borrelidin particles of claim 1 in the preparation of a medicament for the treatment of novel coronavirus pneumonia, said borrelidin particles being capable of significantly reducing the levels of IL-6 secretion and NF- κ Bp65 protein phosphorylation in Calu-3 cells resulting from SARS-CoV-2S protein stimulation.

3. Use of the rarefaction particle of claim 2 for the preparation of a medicament for the treatment of a novel coronavirus pneumonitis, said rarefaction particle modulating the NF- κ B signaling pathway by competitively binding to a SARS-CoV-2 receptor on a cell, to exert its effect against a SARS-CoV-2-induced inflammatory response.