Application of traditional Chinese medicine composition in treatment of influenza viral pneumonia

Abstract

The traditional Chinese medicine composition is prepared from 7.5 g of gypsum, 3 g of herba ephedrae, 4.5 g of fried semen armeniacae amarae, 1.5 g of radix glycyrrhizae, 5 g of cortex magnoliae officinalis, 7.5 g of rhizoma atractylodis fried with bran, 5 g of fried semen tsaoko, 4.5 g of rhizoma pinellinae praeparata, 7.5 g of poria cocos, 5 g of radix astragali, 5 g of semen lepidii, 5 g of radix paeoniae rubra, 5 g of agastache rugosus and 2.5 g of radix et rhizoma rhei.

Description

[0001] Technical field: The present application relates to a Chinese medicine composition for treating influenza virus pneumonia, belonging to the field of Chinese medicine. Background technology:

[0002] Influenza A virus (IAV) belongs to the Orthomyxoviridae family and is a spherical or filamentous single-stranded negative-sense RNA enveloped virus. Influenza caused by influenza A virus is a common acute respiratory infectious disease with epidemic and seasonal characteristics. It has a high morbidity and mortality rate worldwide, posing a serious threat to public health and social economy. According to statistics from the World Health Organization, about 5% to 15% of the world's population is infected with influenza each year, causing 290,000 to 650,000 deaths. Summary of the invention:

[0003] Huashibaidu prescription is composed of 14 herbs including ephedra, patchouli, gypsum, etc. It has the effects of removing dampness and detoxification, promoting lung function and relieving heat, and is used for epidemic diseases caused by dampness and toxins invading the lungs. In order to further expand the application scope of Huashibaidu prescription, the effect and possible mechanism of Huashibaidu prescription on influenza virus pneumonia were studied.

[0004] The Chinese medicine composition involved in the present application is a Chinese medicine prescription for eliminating dampness and eliminating toxins. Specifically, the Chinese medicine composition involved in the present application is: 7.5g gypsum, 3g ephedra, 4.5g stir-fried bitter almonds, 1.5g licorice, 5g Magnolia officinalis, 7.5g stir-fried Atractylodes macrocephala with bran, 5g stir-fried Amomum villosum kernel, 4.5g Pinellia ternata, 7.5g Poria, 5g Astragalus, 5g Lepidium japonicus, 5g Red Peony Root, 5g Patchouli, and 2.5g Rhubarb.

[0005] The preparation method of the Chinese medicine composition is as follows: (1) taking 7.5 g of gypsum, 3 g of ephedra, 4.5 g of fried bitter almond, 1.5 g of liquorice, 5 g of magnolia bark, 7.5 g of fried atractylodes with bran, 5 g of fried grass kernel, 4.5 g of pinellia tuber, 7.5 g of tuckahoe, 5 g of astragalus, 5 g of scutellaria baicalensis, 5 g of red peony root, 5 g of patchouli, and 2.5 g of rhubarb; (2) decocting the gypsum first, and then adding patchouli and rhubarb, adding 5-10 times the amount of water prescribed, decocting for 2-3 times, each time for 0.5-2 hours, filtering, and concentrating the filtrate to a content of 1.71 g crude drug·mL -1 .

[0006] The present application provides the use of the above-mentioned Chinese medicine combination. Specifically, the present application provides an application of a Chinese medicine composition in the preparation of an anti-influenza virus drug, characterized in that the Chinese medicine composition is made of 7.5g gypsum, 3g ephedra, 4.5g stir-fried bitter almonds, 1.5g licorice, 5g magnolia bark, 7.5g stir-fried atractylodes, 5g stir-fried grass kernel, 4.5g pinellia, 7.5g poria, 5g astragalus, 5g scutellaria, 5g red peony root, 5g patchouli, and 2.5g rhubarb.

[0007] The present application provides an application of a traditional Chinese medicine composition in the preparation of a drug for preventing and treating influenza virus pneumonia, characterized in that the traditional Chinese medicine composition is prepared from 7.5 g of gypsum, 3 g of ephedra, 4.5 g of stir-fried bitter almonds, 1.5 g of licorice, 5 g of magnolia bark, 7.5 g of stir-fried atractylodes with bran, 5 g of stir-fried grass kernel, 4.5 g of pinellia, 7.5 g of tuckahoe, 5 g of astragalus, 5 g of scutellaria, 5 g of red peony root, 5 g of patchouli, and 2.5 g of rhubarb.

[0008] In the application, the influenza virus is influenza A virus.

[0009] In the application described herein, the above-mentioned Chinese medicine composition exerts an anti-influenza virus pneumonia effect by inhibiting the expression of key nodes TRAF6 and NFKBIA in the NF-κB signaling pathway, inhibiting the expression of chemokines CCL7, CXCL2, and CCL2, reducing the recruitment of inflammatory cells.

[0010] In the application of the above-mentioned traditional Chinese medicine composition, the prepared medicine is in the form of granules, capsules, tablets, oral liquids, injections and the like. Description of the drawings:

[0011] Figure 1 Lung tissue morphology of mice in each group (H&E, ×200)

[0012] Figure 2 Differential gene analysis, including A and B differential gene volcano plots; C and D differential gene Venn plots

[0013] Figure 3 GO analysis and KEGG analysis of down-regulated core genes, including Figure A GO analysis; Figure B KEGG analysis

[0014] Figure 4 GO enrichment analysis of upregulated core genes

[0015] Figure 5 PPI network analysis of differentially expressed genes Specific implementation method:

[0016] Example 1: Chinese medicine composition and preparation method thereof

[0017] The formula of the Chinese medicine composition is: 7.5g of gypsum, 3g of ephedra, 4.5g of stir-fried bitter almond, 1.5g of licorice, 5g of magnolia bark, 7.5g of stir-fried atractylodes with bran, 5g of stir-fried grass kernel, 4.5g of pinellia, 7.5g of poria, 5g of astragalus, 5g of scutellaria, 5g of red peony root, 5g of patchouli, and 2.5g of rhubarb.

[0018] The preparation method of the Chinese medicine composition is as follows: (1) taking 7.5 g of gypsum, 3 g of ephedra, 4.5 g of fried bitter almond, 1.5 g of liquorice, 5 g of magnolia bark, 7.5 g of fried atractylodes with bran, 5 g of fried grass kernel, 4.5 g of pinellia tuber, 7.5 g of tuckahoe, 5 g of astragalus, 5 g of scutellaria baicalensis, 5 g of red peony root, 5 g of patchouli, and 2.5 g of rhubarb; (2) wherein the gypsum is decocted first, and patchouli and rhubarb are added later, and decocted twice with 8 times the amount of water prescribed, each time for 1 hour, filtered, and the filtrate is concentrated to a content of 1.71 g crude drug·mL -1 .

[0019] Example 2: Pharmacodynamics Experiment

[0020] 2.1 Animals

[0021] Healthy SPF-grade ICR mice, weighing 13-15 g, were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (Animal Production License No.: SCXK (Beijing) 2016-0006). They were kept in an ABSL-2 biosafety room at a temperature of 20-26°C, a humidity of 40-70%, a light-dark cycle of 12 / 12 h, and free access to food. The animal experiments involved in this article were approved by the Animal Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, with approval number 2022D056.

[0022] 2.2 Drugs and reagents

[0023] The experimental Chinese medicine composition is the Chinese medicine composition prepared in Example 1. Influenza A virus mouse lung adapted strain FM1 was purchased from the Institute of Virology, Chinese Academy of Preventive Medicine, and was stored in a -80°C refrigerator for standby use after passage in this laboratory. Isoflurane (Ruipu Biopharmaceutical Co., Ltd., batch number 20200702). Hematoxylin (Beijing Boruijie Technology Co., Ltd., DH0001 / 0405A17). Animal tissue total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., batch number X0708). TransScriptcDNA first-strand synthesis kit, TOP Green fluorescent quantitative PCR kit (Beijing Quanshijin Biotechnology Co., Ltd., batch number P31130, Q20902). NovaSegrm 6000 S4 Cluster Cartridge (Illumina, batch number 20689882).

[0024] 2.3 Instruments

[0025] MC 1.8 biological safety cabinet, Thermo Company. BSA3202S-CW, BSA323S-CW electronic balance, Sartorius Instrument Co., Ltd., AL-204 METTLER TOLEDO electronic balance, Mettler-Toledo Instrument (Shanghai) Co., Ltd. EG 1140H paraffin embedding machine, LEICA819 slicer, Leica, Germany, XL 5010 staining machine, Autostainer, Germany, DM 2500 microscopic digital photography biological microscope Leica (Germany). SCI1000-G PCR amplification instrument (Celo Czech). NanoDrop one micro-volume UV-Vis spectrophotometer (Thermo Company). FQD-96A fluorescence quantitative polymerase chain reaction (Hangzhou Bio-Ri Technology Co., Ltd.). Agilent 2100 Bioanalyzer biological analyzer. Noveseq 6000 sequencing (Illumina Company).

[0026] 3 Methods

[0027] 3.1 Effect of Huashibaidu prescription on death of mice caused by influenza virus infection

[0028] A total of 90 SPF ICR mice weighing 13-15 g were randomly divided into normal group (10 mice), model group, high-, medium-, and low-dose groups of Huashi Baidu Formula (17.81, 8.91, and 4.45 g kg -1 ), 20 mice in each group, half male and half female. Modeling: Except for the normal group, the mice in the other groups were anesthetized by isoflurane inhalation, and the mice were infected with influenza virus mouse lung adapted strain FM1 solution by intranasal drops (0.05mL / mouse), and the normal group was intranasally dropped with an equal volume of saline. Administration: On the day of infection, each group was given oral administration, and the normal group and the model group were given the same volume of pure water (20mL·kg -1 ), once a day for 5 consecutive days. The death of mice was observed within 14 days, the survival time of mice was recorded, and the survival time extension rate was calculated according to the following formula.

[0029] Survival time extension rate = (survival time of drug administration group - survival time of model group) / survival time of model group × 100%

[0030] 3.2 Effect of Huashibaidu Recipe on Influenza Virus Pneumonia

[0031] 3.2.1 Modeling, grouping and drug administration

[0032] Grouping: 60 healthy SPF ICR mice weighing 13-15 g were randomly divided into normal group, model group, high-dose, medium-dose and low-dose groups of Huashi Baidu Formula (17.81, 8.91, 4.45 g kg -1), 12 mice in each group, half male and half female. Modeling: Except for the normal group, the mice in the other groups were anesthetized by isoflurane inhalation, and the mice were infected with influenza virus mouse lung adapted strain FM1 solution by intranasal drops (0.05mL / mouse), and the normal group was intranasally dropped with an equal volume of normal saline. Drug administration: On the day of infection, each group was given oral administration, and the normal group and model group were given the same volume of pure water (20mL·kg -1 body weight), once a day, for 5 consecutive days.

[0033] 3.2.2 Effect of Huashi Baidu Recipe on Lung Index of Mice with Influenza Virus Pneumonia

[0034] On the 6th day after virus infection, mice were weighed, killed, fixed, dissected, lungs were removed, lung weights were weighed, and lung index and lung index inhibition rate were calculated according to the following formula.

[0035] Lung index = lung weight / body weight × 100

[0036] Lung index inhibition rate = (lung index value of model group - lung index value of drug administration group) / (lung index value of model group - lung index value of normal group) × 100%

[0037] 3.2.3 Effect of Huashi Baidu Recipe on the pathological morphology of lung tissue in mice with influenza virus pneumonia

[0038] On the 6th day of virus infection, the left lung of the mouse was fixed in 10% formaldehyde solution, taken out after 48 hours, rinsed with running water, dehydrated with gradient ethanol, transparentized with xylene, embedded in paraffin, sliced, stained with H&E, sealed with neutral gum, observed under a microscope, and pathological histological examination was performed. The evaluation criteria are as follows:

[0039] “-” There was no inflammatory exudation or blood stasis in the interstitial lung tissue of mice, and the tissue structure was normal.

[0040] There was no obvious large-scale exudative inflammation in the interstitial lung tissue of “+” mice, but there was a small amount of exudative exudate in the interstitium.

[0041] “++” mice had localized inflammatory exudation in the interstitial lung tissue, and edema and congestion in the interstitial blood vessels.

[0042] “+++” mice had large areas of inflammatory exudates in the lung interstitium, mainly lymphocytes, with a small number of eosinophils, monocytes, and macrophages, and congestion in the pulmonary interstitial blood vessels.

[0043] "++++" mice had extensive hemorrhage and inflammatory exudation in the lung tissue interstitium, cell proliferation and hypertrophy, and increased lymphocytes. A small amount of eosinophilic exudation, and eosinophilic changes in small blood vessels. The lesions were diffuse and severe, such as displacement of intimal cells, incomplete cells, and swelling of the tissue interstitium. Inflammation around the bronchioles, with sloughing in the cavity.

[0044] 3.2.4 Effect of Huashi Baidu Recipe on Viral Load in Lung Tissue of Mice Infected with Influenza Virus

[0045] The right lung tissues of mice in each group were collected, and the qRT-PCR absolute quantitative method was used to detect the viral load of influenza virus in the lung tissues. The primer sequences used are shown in Table 1.

[0046] Table 1 Primer sequences

[0047]

[0048] 3.3 Exploration of the mechanism of action of Huashibaidu prescription in improving influenza virus pneumonia based on transcriptomics

[0049] 3.3.1 Total RNA extraction from lung tissue and cDNA library construction

[0050] On the 6th day after virus infection, lung tissues of mice in the normal group, model group, and high-dose HSDPD group were collected, and total RNA was extracted using RNAEasy Fast tissue / Cell Kit. The concentration and purity of the extracted RNA were detected by Nanodrop2000, the integrity of RNA was detected by agarose gel electrophoresis, and the RIN value was determined by Agilent2100.

[0051] The main process of constructing an mRNA library is as follows: (1) using magnetic beads with Oligo(dT) to separate mRNA; (2) randomly breaking the enriched mRNA into small fragments of about 300 bp; (3) using random primers to reverse the broken mRNA to generate double-linked cDNA; (4) repairing the ends of the double-linked cDNA, adding "A" bases and connecting "Y" adapters; (5) amplifying the sequencing library and purifying the cDNA library; (6) dsDNA System quantitative library; (7) The library was sequenced using the Illumina Novaseq6000 sequencing platform.

[0052] 3.3.2 Differential gene screening and functional enrichment analysis

[0053] The obtained raw data were quality controlled, and low-quality bases containing adapters were removed to obtain high-quality sequencing sequences (clean reads) for transcriptome analysis. The clean reads were compared with the reference genome using HISAT2 software; the expression levels of genes were quantitatively analyzed using RSEM software; and the significance of differential gene expression was analyzed using DESeq2 software. The default screening criteria for significantly differentially expressed genes were: FDR < 0.05 & |log2FC| ≥ 1.

[0054] The up-regulated genes in the model group vs the normal group and the down-regulated genes in the Huashi Baidu Formula vs the model group, as well as the down-regulated genes in the model group vs the normal group and the up-regulated genes in the Huashi Baidu Formula vs the model group were analyzed to screen out the down-regulated and up-regulated core genes of the Huashi Baidu Formula against influenza virus pneumonia; the GO functional enrichment analysis and KEGG PATHWAY enrichment analysis of the screened core genes were performed using the Metascape website (https: / / metascape.org / gp / index.html# / main / step1). The test method was Fisher's exact test.

[0055] 3.3.3 PPI analysis and verification of core genes

[0056] For the down-regulated core genes, PPI analysis was performed using the STRING website (https: / / cn.string-db.org / ), and the top five core genes with combined scores were screened out, namely TRAF6, NFKBIA, CCL2, CCL7, and CXCL2.

[0057] The above core genes were verified by qRT-PCR method, and total RNA from lung tissue was extracted by RNA Easy Fast Tissue / CellKit RNA Easy Fast kit; the concentration and purity of the extracted RNA were detected by Nanodrop2000; First-stand cDNA Synthesis SuperMix Kit was used for cDNA reverse transcription. All the above experimental operations were carried out according to the product instructions. Amplification was performed under the following conditions: pre-denaturation at 94℃ for 30s; denaturation at 94℃ for 5s, annealing at 60℃ for 40s, and extension for 40s. -ΔΔCt The relative expression of genes in different treatment groups was calculated by the primer sequence shown in Table 2.

[0058] Table 2 Primer sequence list

[0059]

[0060] 3.4 Statistical analysis

[0061] SPSS 21.0 was used to perform statistical analysis on the data. The SW test was used to verify the normality of the measurement data, and the Levene test was used to verify the homogeneity of variance. When P>0.05, the data were considered to have normality and homogeneity of variance. The data were compared with the Kruskal-Wallis rank sum test for pathological grade data. P < 0.05 indicated that the difference was statistically significant.

[0062] 4 Results

[0063] 4.1 Protective effect of Huashi Baidu prescription on mortality of mice with influenza virus pneumonia

[0064] 14 days after influenza virus infection, all mice in the model group died, with a mortality rate of 100%. Compared with the model group, the survival time of the high-dose Huashi Baidu Recipe group was significantly prolonged (P < 0.05). The results are shown in Table 3.

[0065] Table 3 Effect of Huashibaidu prescription on mortality of mice with influenza virus pneumonia

[0066]

[0067] Note: Compared with the normal group 1) P<0.01; compared with the model group 2) P<0.05

[0068] 4.2 Effect of Huashibaidu prescription on the body weight and lung index of mice with influenza virus pneumonia

[0069] After being infected with influenza virus, the body weight of mice in the model group was significantly reduced (P<0.01) and the lung index was significantly increased (P<0.01) compared with the normal group; compared with the model group, the lung index values ​​of the high, medium and low dose groups of Huashi Baidu Fang were reduced to varying degrees, and the high dose group was significantly reduced (P<0.05). The results are shown in Table 4.

[0070] Table 4 Effect of Huashibaidu prescription on lung index of influenza virus pneumonia in mice

[0071]

[0072] Note: Compared with the normal group 1) P<0.01; compared with the model group 2) P<0.05

[0073] 4.3 Effect of Huashibaidu prescription on the pathological morphology of mice with influenza virus pneumonia

[0074] The interstitial lung tissue of mice in the normal group was not widened, and no inflammation, exudation, edema and other lesions were found in the interstitium; the alveoli were not enlarged, and no inflammatory hyperplasia was found around the bronchioles in the lungs, and the tissue structure was normal. The alveolar septa of the mice in the model group were significantly widened, and the inflammatory cells and lymphocytes were significantly increased, and a small amount of inflammatory cells such as monocytes and macrophages were exuded; there was obvious congestion in the small blood vessels of the lungs, and some vascular intima fell off; there was swelling in the interstitium of the lungs, and there was obvious inflammation around some bronchioles in the lungs, mainly lymphocytes, and the endothelium of the bronchioles fell off, and inflammatory cells replaced. Compared with the model group, the lesions of bronchioles, interstitial inflammation, congestion, swelling and other lesions in the lung tissues of mice in the high, medium and low dose groups of the Huashi Baidu prescription group were alleviated to varying degrees. The results of the rank sum test showed that there were significant differences compared with the model group.

[0075] Results Figure 1 , Table 5.

[0076] Table 5 Effect of Huashi Baidu Fang on lung lesions in mice with viral pneumonia

[0077]

[0078] Note: The P value of the rank sum test is the comparison between each group and the model control group

[0079] 4.4 Huashibaidu prescription reduces the viral load in lung tissue of mice with influenza virus pneumonia

[0080] After mice were infected with influenza A virus, the viral load in the lung tissue of the model group mice was significantly increased compared with the normal group (P < 0.05); compared with the model group, the viral load in the lung tissue of the high-dose and medium-dose groups of Huashi Baidu Fang mice was reduced to varying degrees. The results are shown in Table 6.

[0081] Table 6 Effect of Huashi Baidu Recipe on viral load of viral pneumonia in mice

[0082]

[0083] Note: Compared with the normal group 1) P<0.05

[0084] 4.5 GO and KEGG enrichment analysis of differentially expressed genes

[0085] After the intervention of Huashibaidu prescription, 81 down-regulated core genes and 232 up-regulated core genes were screened (see Figure 2). GO functional enrichment analysis of down-regulated core genes showed that differentially expressed genes (DEGs) were mainly enriched in cytokine-mediated signaling pathways, chemokine activity, chemokine receptor binding, etc.; KEGG enrichment analysis showed that they were mainly enriched in IL-17 signaling pathway, NF-κB signaling pathway, cytokine-cytokine receptor interaction, etc. GO enrichment analysis of up-regulated core genes showed that DEGs were mainly enriched in DNA-binding transcription repressor activity, Cul3-RING ubiquitin ligase complex, calcium ion binding, etc.; KEGG enrichment analysis obtained Herpes simplex virus 1 infection, see Figure 3 , 4 .

[0086] 4.6 PPI analysis results of core genes

[0087] PPI analysis was performed on the down-regulated core genes. The top five node genes with the combined score were TRAF6, NFKBIA, CCL2, CCL7, and CXCL2. TRAF6 and NFKBIA are two key genes in the NF-κB signaling pathway, and CCL2, CCL7, and CXCL2 are three key genes in the cytokine-cytokine receptor interaction. Figure 5 And Table 7.

[0088] Table 7 Node relationship table of some protein interaction network analysis

[0089]

[0090] 4.7 Verification of down-regulated core genes in the Huashi Baidu Formula against influenza virus pneumonia

[0091] Compared with the normal group, the relative mRNA expression of TRAF6, NFKBIA, CXCL2, CCL7, and CCL2 in the model group increased (P < 0.01); compared with the model group, the relative mRNA expression of TRAF6, NFKBIA, CXCL2, CCL7, and CCL2 in the high-dose group of Huashi Baidu Fang decreased (P < 0.01 or P < 0.05), and the relative mRNA expression of NFKBIA, CXCL2, and CCL7 in the medium-dose group of Huashi Baidu Fang decreased (P < 0.01 or P < 0.05). The results are shown in Table 8.

[0092] Table 8 Relative mRNA expression in each group ( n=6)

[0093]

[0094] Note: Compared with the normal group 1) P<0.01; compared with the model group 2) P<0.05, 3) P<0.01

Claims

1. Use of a Chinese medicine composition in the preparation of an anti-influenza virus drug, characterized in that: The Chinese medicine composition is prepared from 7.5g of gypsum, 3g of ephedra, 4.5g of stir-fried bitter almonds, 1.5g of licorice, 5g of magnolia bark, 7.5g of stir-fried atractylodes with bran, 5g of stir-fried grass kernel, 4.5g of pinellia, 7.5g of tuckahoe, 5g of astragalus, 5g of scutellaria, 5g of red peony root, 5g of patchouli, and 2.5g of rhubarb.

2. Use of a Chinese medicine composition in the preparation of a drug for anti-influenza virus pneumonia, characterized in that: The Chinese medicine composition is prepared from 7.5g of gypsum, 3g of ephedra, 4.5g of stir-fried bitter almonds, 1.5g of licorice, 5g of magnolia bark, 7.5g of stir-fried atractylodes with bran, 5g of stir-fried grass kernel, 4.5g of pinellia, 7.5g of tuckahoe, 5g of astragalus, 5g of scutellaria, 5g of red peony root, 5g of patchouli, and 2.5g of rhubarb.

3. The use according to claim 1 or claim 2, wherein: The preparation method of the Chinese medicine composition is as follows: (1) taking 7.5 g of gypsum, 3 g of ephedra, 4.5 g of fried bitter almond, 1.5 g of liquorice, 5 g of magnolia bark, 7.5 g of fried atractylodes with bran, 5 g of fried grass kernel, 4.5 g of pinellia tuber, 7.5 g of tuckahoe, 5 g of astragalus, 5 g of scutellaria baicalensis, 5 g of red peony root, 5 g of patchouli, and 2.5 g of rhubarb; (2) decocting the gypsum first, and then adding patchouli and rhubarb, adding 5-10 times the amount of water prescribed, decocting for 2-3 times, each time for 0.5-2 hours, filtering, and concentrating the filtrate to a content of 1.71 g crude drug·mL -1 .

4. The use according to claim 1 or claim 2, wherein the influenza virus is influenza A virus.

5. The use according to claim 1 or claim 2, wherein: The Chinese medicine composition inhibits the expression of key nodes TRAF6 and NFKBIA of the NF-κB signaling pathway, inhibits the expression of chemokines CCL7, CXCL2, and CCL2, reduces the recruitment of inflammatory cells, and exerts an anti-influenza virus pneumonia effect.

6. The use according to claim 1 or claim 2, wherein: The prepared medicine is in the form of granules, capsules, tablets, oral liquids and injections.

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