Use of a traditional Chinese medicine composition for treating influenza and influenza-related bacterial infections in the preparation of a medicine for treating influenza-related bacterial infections

Abstract

The present invention relates to a Chinese medicine composition for treating influenza and influenza-induced bacterial infections. The composition is prepared from the following raw materials in parts by weight: 3-15 parts of honeysuckle, 3-15 parts of schizonepeta, 6-18 parts of astragalus, 3-15 parts of patchouli, 3-15 parts of siler, 3-15 parts of isatis root, 3-15 parts of platycodon, 9-21 parts of phragmites, and 2-8 parts of liquorice. The present invention has been confirmed through animal experiments that the prescription has a good in vivo protective effect on influenza and influenza-induced secondary infection mice, can significantly alleviate inflammatory reactions and organ damage, and at the same time, the medicinal flavors in the composition of the present invention have the advantages of wide source, safety, low toxicity, low cost, etc., are very suitable for large-scale promotion and application, and have obvious social benefits and economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to application of Jingyin Gubiao prescription in treating influenza and bacterial infection diseases secondary to influenza. Background Art

[0002] Unlike the common cold, influenza is epidemiologically characterized by sudden outbreaks and rapid spread, resulting in varying degrees of epidemic. Clinically, influenza presents with an acute onset and severe symptoms, manifesting as systemic toxicity such as high fever, headache, fatigue, and muscle aches. Respiratory symptoms are mild or subtle, and complications are common. Complications such as pneumonia, encephalitis, myocarditis, myositis, and acute myocardial infarction may also occur. Influenza virus pneumonia, in particular, has a high incidence rate and is the most common form of community-acquired viral pneumonia, contributing significantly to its high mortality rate. Post-influenza bacterial infection occurs after the virus has cleared, resulting in susceptibility to bacteria caused by the viral infection. Influenza virus-associated bacterial pneumonia, unlike so-called community-acquired pneumonia, is most commonly caused by Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae. Co-infection with Staphylococcus aureus is a hallmark of influenza virus pneumonia.

[0003] The treatment of influenza and secondary bacterial infections of influenza mainly includes antiviral and antibacterial treatments. Many drugs have been developed. Among them, Western medicine is quick to take effect but also has side effects. Therefore, traditional Chinese medicine has gradually become an important choice in the treatment plan. There are traditional Chinese medicine formulas for treating influenza, such as Chinese document CN116870078A, published on October 13, 2023, which discloses a traditional Chinese medicine composition for treating influenza and its method, composition particles and application. The traditional Chinese medicine composition includes the following components: honeysuckle, forsythia, schizonepeta, polygonum indigo leaf, cotton The Chinese medicine composition for treating influenza provided by the invention, including Rhizoma Cynanchum, fried Burdock Fructus, Belamcanda, Scrophulariaceae, and Rhubarb, has a significant antipyretic effect on yeast and endotoxin fever models, a significant inhibitory effect on acute inflammation caused by chemical inflammatory agents, and can significantly reduce the number of writhing times, prolong the pain threshold of mice, have a significant analgesic effect, and have a significant antitussive and expectorant effect on mice, that is, the Chinese medicine composition described in this application can simultaneously play the role of antipyretic, anti-inflammatory, antitussive and analgesic, and can simultaneously relieve various symptoms of influenza, and has a good therapeutic effect on influenza. There are also Chinese medicine formulas for treating secondary bacterial infections of influenza, such as the Chinese document CN110420234A, published on November 8, 2019, which discloses the use of Babaodan in the preparation of a drug for preventing or treating secondary bacterial infections of influenza, but the Chinese medicine formula for treating influenza and secondary bacterial infections of influenza as described in this application has not been reported. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies in the prior art and provide an application of Jingyin Gubiao prescription in treating influenza and influenza-related secondary bacterial infections.

[0005] On the one hand, the present invention provides a traditional Chinese medicine composition for treating influenza and secondary bacterial infections caused by influenza, which is prepared from the following raw materials in parts by weight: 3-15 parts of honeysuckle, 3-15 parts of schizonepeta, 6-18 parts of astragalus, 3-15 parts of patchouli, 3-15 parts of siler, 3-15 parts of isatis root, 3-15 parts of platycodon, 9-21 parts of reed rhizome, and 2-8 parts of licorice.

[0006] As a preferred example, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 6-12 parts of honeysuckle, 6-12 parts of schizonepeta, 9-15 parts of astragalus, 6-12 parts of patchouli, 6-12 parts of siler, 6-12 parts of isatis root, 6-12 parts of platycodon, 12-18 parts of reed rhizome, and 4-8 parts of licorice.

[0007] More preferably, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 9 parts of honeysuckle, 9 parts of schizonepeta, 12 parts of astragalus, 9 parts of patchouli, 9 parts of siler, 9 parts of isatis root, 9 parts of platycodon, 15 parts of reed rhizome, and 6 parts of liquorice.

[0008] In a second aspect, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned traditional Chinese medicine compositions.

[0009] As a preferred example, the dosage form of the drug is selected from any one of tea, granules, syrup, capsules, oral preparations, tablets, pills, and vesicles.

[0010] In a third aspect, the present invention provides use of any of the above-mentioned traditional Chinese medicine compositions or any of the pharmaceutical compositions in the preparation of a drug for treating influenza.

[0011] In a fourth aspect, the present invention further provides use of any of the above-mentioned traditional Chinese medicine compositions or any of the above-mentioned pharmaceutical compositions in the preparation of treatments for secondary bacterial infections of influenza. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Attachment Figure 1 The effect of Jingyin Gubiao prescription on weight protection and survival of influenza-infected mice; A: 14-day survival of influenza-infected mice; B: 14-day weight changes of influenza-infected mice.

[0013] Attachment Figure 2 The effects of Jingyin Gubiao prescription on the body weight and lung index of influenza-infected mice; A: Body weight changes after 6 consecutive days of drug administration in mice infected with influenza virus; B: Lung index 6 days after severe influenza virus model in mice; C: Lung pathological changes 6 days after severe influenza virus model in mice.

[0014] Attachment Figure 3 Effects of Jingyin Gubiao Recipe on symptoms and survival of mice secondary to influenza infection; A: Symptom scores of mice secondary to influenza infection on days 6 to 9; B: Survival of mice secondary to influenza infection on day 19.

[0015] Attachment Figure 4 The effects of high- and low-dose groups of Jingyin Gubiao prescription on the body weight and lung index of mice secondary to influenza infection; A: Weight changes of mice 6 days after secondary infection with Staphylococcus aureus by influenza virus; B: Changes in lung index of mice after secondary infection with Staphylococcus aureus by influenza virus.

[0016] Attachment Figure 5 HE staining images of lung and liver tissues of mice in each group 6 days after infection.

[0017] Attachment Figure 6 This is the effect of high-dose and low-dose groups of Jingyin Gubiao prescription on the expression of inflammatory factor proteins in the lungs of mice secondary to influenza infection; AF represents the expression of inflammatory factors TNF-α, IL-6, CCL5, MIP-2, ICAM1 and IL-10 proteins in the lungs on the 6th day of infection after continuous administration for 6 days.

[0018] Attachment Figure 7 This is the effect of high-dose and low-dose groups of Jingyin Gubiao prescription on the mRNA expression levels of lung inflammatory factors in mice secondary to influenza infection; AD represents the expression of lung inflammatory factors TNF-α, CCL5, MIP-2, and MCP-1 mRNA on the 6th day of infection after continuous administration for 6 days.

[0019] Attachment Figure 8 This is the effect of high-dose and low-dose groups of Jingyin Gubiao prescription on the expression levels of serum inflammatory factor proteins in mice with secondary influenza infection; AC represents the expression of serum inflammatory factors IL-6, TNF-α, and IFN-γ proteins on the 6th day of infection after continuous administration for 6 days. DETAILED DESCRIPTION

[0020] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention record, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the application's appended claims.

[0021] Example 1 Traditional Chinese medicine composition for treating influenza and influenza-related bacterial infections according to the present invention (I)

[0022] 9 parts of Flos Honeysuckle, 9 parts of Herba Schizonepetae, 12 parts of Radix Astragali, 9 parts of Herba Patchouli, 9 parts of Saposhnikoviae, 9 parts of Radix Isatidis, 9 parts of Platycodon, 15 parts of Rhizoma Phragmites, 6 parts of Radix Glycyrrhizae.

[0023] Example 2 Traditional Chinese medicine composition for treating influenza and influenza-related bacterial infections (II)

[0024] 9 parts of Flos Honeysuckle, 6 parts of Herba Schizonepetae, 15 parts of Radix Astragali, 3 parts of Herba Patchouli, 15 parts of Saposhnikoviae, 9 parts of Radix Isatidis, 6 parts of Platycodon grandiflorum, 18 parts of Rhizoma Phragmites, and 2 parts of Radix Glycyrrhizae.

[0025] Example 3 Traditional Chinese medicine composition for treating influenza and influenza-related bacterial infections (III)

[0026] 6 parts of Flos Honeysuckle, 12 parts of Herba Schizonepetae, 6 parts of Radix Astragali, 15 parts of Herba Patchouli, 9 parts of Saposhnikoviae, 6 parts of Radix Isatidis, 12 parts of Platycodon, 9 parts of Rhizoma Phragmites, and 10 parts of Radix Glycyrrhizae.

[0027] Example 4 Traditional Chinese medicine composition for treating influenza and influenza-related bacterial infections (IV)

[0028] 12 parts of Flos Honeysuckle, 3 parts of Herba Schizonepetae, 18 parts of Radix Astragali, 9 parts of Herba Patchouli, 6 parts of Saposhnikoviae, 12 parts of Radix Isatidis, 3 parts of Platycodon, 21 parts of Rhizoma Phragmites, and 6 parts of Radix Glycyrrhizae.

[0029] Example 5 Traditional Chinese medicine composition for treating influenza and influenza-related bacterial infections (V)

[0030] 3 parts of Flos Honeysuckle, 15 parts of Herba Schizonepetae, 12 parts of Radix Astragali, 6 parts of Herba Patchouli, 12 parts of Saposhnikoviae, 3 parts of Radix Isatidis, 15 parts of Platycodon, 15 parts of Rhizoma Phragmites, and 4 parts of Radix Glycyrrhizae.

[0031] Example 6 Traditional Chinese medicine composition for treating influenza and influenza-related bacterial infections (VI)

[0032] 15 parts of Flos Honeysuckle, 9 parts of Herba Schizonepetae, 9 parts of Radix Astragali, 12 parts of Herba Patchouli, 3 parts of Saposhnikoviae, 15 parts of Radix Isatidis, 9 parts of Platycodon grandiflorum, 12 parts of Rhizoma Phragmites, 8 parts of Radix Glycyrrhizae.

[0033] Example 7 Traditional Chinese Medicine Composition for Treating Influenza and Bacterial Infections Following Influenza (VII)

[0034] 9 parts of Flos Honeysuckle, 12 parts of Herba Schizonepetae, 6 parts of Radix Astragali, 15 parts of Herba Patchouli, 9 parts of Saposhnikoviae, 6 parts of Radix Isatidis, 12 parts of Platycodon, 9 parts of Rhizoma Phragmites, and 10 parts of Radix Glycyrrhizae.

[0035] Example 8 Traditional Chinese medicine composition for treating influenza and influenza-related bacterial infections (VIII)

[0036] 6 parts of Flos Honeysuckle, 3 parts of Herba Schizonepetae, 18 parts of Radix Astragali, 9 parts of Herba Patchouli, 6 parts of Radix Saposhnikoviae, 12 parts of Radix Isatidis, 3 parts of Platycodon grandiflorum, 21 parts of Rhizoma Phragmites, and 6 parts of Radix Glycyrrhizae.

[0037] Example 9 Traditional Chinese Medicine Composition for Treating Influenza and Bacterial Infections Following Influenza (IX)

[0038] 12 parts of Flos Honeysuckle, 15 parts of Herba Schizonepetae, 12 parts of Radix Astragali, 6 parts of Herba Patchouli, 12 parts of Saposhnikoviae, 3 parts of Radix Isatidis, 15 parts of Platycodon grandiflorum, 15 parts of Rhizoma Phragmites, and 4 parts of Radix Glycyrrhizae.

[0039] Example 10 Traditional Chinese Medicine Composition for Treating Influenza and Bacterial Infections Following Influenza (Ten)

[0040] 3 parts of Flos Honeysuckle, 9 parts of Herba Schizonepetae, 9 parts of Radix Astragali, 12 parts of Herba Patchouli, 3 parts of Saposhnikoviae, 15 parts of Radix Isatidis, 9 parts of Platycodon grandiflorum, 12 parts of Rhizoma Phragmites, and 8 parts of Radix Glycyrrhizae.

[0041] Example 11 Traditional Chinese Medicine Composition for Treating Influenza and Bacterial Infections Following Influenza (XI)

[0042] 15 parts of Flos Honeysuckle, 6 parts of Herba Schizonepetae, 15 parts of Radix Astragali, 3 parts of Herba Patchouli, 15 parts of Saposhnikoviae, 9 parts of Radix Isatidis, 6 parts of Platycodon grandiflorum, 18 parts of Rhizoma Phragmites, and 2 parts of Radix Glycyrrhizae.

[0043] Example 12 Effects of Jingyin Gubiao Recipe on Weight Protection and Survival of Influenza Mice

[0044] 1. Drug preparation

[0045] Jingyin Gubiao Fang is Example 1 of the present invention. Jingyin Gubiao Fang granules are prepared and provided by Shanghai Wanshicheng Chinese Medicine Products Co., Ltd. The daily clinical dose of the granules is 14g / person / day (adults). The drug dosage is converted according to the kilogram body weight of humans and mice. The weight of an adult is 60kg. The clinical dosage for humans is 0.233g / kg, and the dosage for mice is 12 times the human dosage, that is, 2.8g / kg. The drug concentration for oral gavage to mice is 0.28g / mL.

[0046] One capsule of oseltamivir (OSV) contains 75 mg of drug, which is dissolved in 25 mL of mineral water to a final concentration of 3 mg / mL. The daily dose for mice is 30 mg / kg, that is, the daily dose for a 20 g mouse is 0.2 mL.

[0047] 2 Experimental animals and virus strains

[0048] Female, SPF-qualified C57BL / 6J mice, 6 weeks old, weighing 17–20 g, were purchased from the joint venture, SIP-BK Laboratory Animal Co., Ltd. (Shanghai, China). They were housed in a constant temperature (23 ± 2°C), relative humidity (60%–65%), and automatic light–dark cycle (LD 12:12, i.e., 7:00–19:00 light, 19:00–7:00 dark). The animals had free access to food, water, and movement. Mice were acclimated for one week before the experiments. All experiments were conducted in a biosafety level 2 (BSL-2) laboratory approved by the Animal Welfare and Ethics Committee of the Institut Pasteur of the Chinese Academy of Sciences in Shanghai, with the experimental number A2021-L027.

[0049] The mouse-adapted influenza virus strain (influenzavirus A / Puerto Rico / 08 / 1934, PR8) was kindly donated by Professor Xu Ke from the Shanghai Pasteur Institute of the Chinese Academy of Sciences. It was amplified in canine kidney cells (MDCK) by our laboratory and the TCID 50 Aliquot and freeze in liquid nitrogen. Use the aliquoted PR8 virus fresh each time to avoid repeated freezing and thawing.

[0050] 3 Experimental methods

[0051] After one week of adaptive feeding, C57BL / 6J mice (7 weeks of age) were randomly divided into a blank control (NC) group, an influenza virus PR8 infection group, a PR8+OSV treatment group, and a PR8+Jingyin Gubiao Formula (JYGBF) treatment group. The JYGBF group received a clinically equivalent dose of 0.233 g / kg, equivalent to a 2.8 g / kg dose for mice; the oseltamivir group received 30 mg / kg daily.

[0052] On the day of mouse infection, the aliquoted influenza virus stock solution was serially diluted with sterile, cold PBS to the experimental concentration and stored on ice water until use. Mice were anesthetized using an anesthesia machine. Once anesthesia was complete, 50 μL of the diluted virus solution was pipetted dropwise through one nostril, allowing the virus to enter the lungs with the mouse's breath, thus establishing a mouse model of influenza virus-induced pneumonia.

[0053] After successful infection, mice were weighed and dosed within 2 hours for each group. For the next 7 consecutive days, drug administration and weight recording were performed. From day 8 to day 14, mice were weighed but no drug administration was performed. A weight loss exceeding 25% was defined as a humane endpoint, and mice were sacrificed. Line graphs of mouse weight changes and survival curves were plotted, and statistical differences between groups were calculated.

[0054] 4. Statistics and Analysis

[0055] Data are expressed as mean ± standard deviation (SD). Multiple comparisons were performed using one-way analysis of variance (Bonferroni test and Dunnett's test). Survival functions were estimated using the Kaplan-Meier method, and differences in survival distributions between groups were compared using the log-rank test. P < 0.05, P < 0.01, or P < 0.001 indicated statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).

[0056] 5 Results

[0057] See the results Figure 1 , Figure 1 A is to establish a mouse influenza virus infection model and then administer the drugs for 7 consecutive days, and observe and record the survival of the mice for 14 days. Figure 1 A shows that the survival rate of mice in the PR8 group was 30.7%; the survival rate in the OSV group was 100%; and the survival rate of the Jingyin Gubiao formula was approximately 92.3%. Compared with the PR8 infection model group, the survival rate of influenza mice in the drug-treated group was significantly improved, and both were statistically significant (*P<0.05). Figure 1 B is to establish a severe influenza virus model in mice and then administer the drugs for 7 consecutive days, and observe and record the weight changes of the mice for 14 days. Figure 1 As shown in Figure 2, the PR8 group showed a significant weight loss trend compared to the blank group. The PR8+OSV and PR8+JYGBF treatment groups significantly improved the weight loss trend in influenza mice (*P<0.05). These results demonstrate that the Jingyin Gubiao formula protects against weight loss and survival in influenza mice.

[0058] Example 13 Jingyin Gubiao Recipe Alleviates Weight Loss and Reduces Lung Index in Influenza-Infected Mice and Protects Lung Tissue Organ Damage Induced by Influenza Virus

[0059] 1. Drug preparation

[0060] Same as Example 12.

[0061] 2 Experimental animals and virus strains

[0062] Same as Example 12.

[0063] 2.3 Experimental methods

[0064] After one week of adaptive feeding, C57BL / 6J mice (7 weeks of age) were randomly divided into a blank control (NC) group, an influenza virus PR8 infection group, a PR8+OSV treatment group, and a PR8+Jingyin Gubiao Formula (JYGBF) treatment group. The JYGBF group received a clinically equivalent dose of 0.233 g / kg, equivalent to a 2.8 g / kg dose for mice; the oseltamivir group received 30 mg / kg daily.

[0065] On the day of mouse infection, the aliquoted influenza virus stock solution was serially diluted with sterile, cold PBS to the experimental concentration and stored on ice water until use. Mice were anesthetized using an anesthesia machine. Once anesthesia was complete, 50 μL of the diluted virus solution was pipetted dropwise through one nostril, allowing the virus to enter the lungs with the mouse's breath, thus establishing a mouse model of influenza virus-induced pneumonia.

[0066] After successful infection, the mice were weighed and the drug administration was completed within 2 hours. The mice were administered and their weights were recorded for 6 consecutive days. On the 6th day after modeling, the lung tissue of the mice was obtained, the organ weight was weighed, and the lung index was calculated. Lung tissues were taken for hematoxylin-eosin staining (HE staining) to observe the protective effect of JYGBF on the lungs of influenza-infected mice.

[0067] 4. Statistics and Analysis

[0068] Same as Example 12

[0069] 5 Results

[0070] See the results Figure 2 , Figure 2 A shows the weight changes of mice after the influenza virus infection model was established and the mice were given drugs for 6 consecutive days. Figure 2 As shown in A, the PR8 group showed a significant weight loss trend compared with the blank group. The PR8+OSV treatment group and the PR8+JYGBF treatment group could significantly improve the weight loss trend of influenza mice and were statistically significant (*P<0.05); Figure 2 B shows the lung index of influenza mice on the 6th day of infection after the establishment of a severe influenza virus model in mice. The drugs were administered for 6 consecutive days. As shown in the figure, after infection with influenza virus PR8, the lung index of mice increased significantly (*P<0.05), indicating that the lungs may suffer from pulmonary edema, hemorrhage and other pathological damage after influenza virus infection. The OSV treatment group and the JYGBF treatment group can significantly reduce the lung index of influenza mice (*P<0.05). This shows that JYGBF can reduce the lung index of influenza mice and alleviate lung damage. Figure 2 C demonstrates the protective effect of JYGBF on lung damage in influenza-infected mice. As shown in the figure, after influenza virus infection, the alveolar structure of mice was destroyed, the infiltration of inflammatory cells and mediators in the lungs increased, bleeding increased, and the lung interstitium thickened with a large number of lymphocytes infiltrate. After OSV and JYGBF treatment, the alveolar tissue of mice was protected, and inflammatory cell infiltration was significantly reduced. These results demonstrate that the Jingyin Gubiao formula alleviates weight loss in influenza-infected mice, reduces the lung index of influenza-infected mice, and protects against influenza virus-induced lung tissue and organ damage.

[0071] Example 14 Effects of Jingyin Gubiao Recipe on Symptom Relief and Survival Protection of Mice Secondarily Infected with Influenza 1. Drug Preparation

[0072] Same as Example 12.

[0073] 2 Experimental animals and virus strains

[0074] Female, SPF-qualified C57BL / 6J mice, 6 weeks old, weighing 17–20 g, were purchased from the joint venture, SIP-BK Laboratory Animal Co., Ltd. (Shanghai, China). They were housed in a constant temperature (23 ± 2°C), relative humidity (60%–65%), and automatic light–dark cycle (LD 12:12, i.e., 7:00–19:00 light, 19:00–7:00 dark). The animals had free access to food, water, and movement. Mice were acclimated for one week before the experiments. All experiments were conducted in a biosafety level 2 (BSL-2) laboratory approved by the Animal Welfare and Ethics Committee of the Institut Pasteur of the Chinese Academy of Sciences in Shanghai, with the experimental number A2021-L027.

[0075] The mouse-adapted influenza virus strain (influenzavirus A / Puerto Rico / 08 / 1934, PR8) was kindly donated by Professor Xu Ke from the Shanghai Pasteur Institute of the Chinese Academy of Sciences. It was amplified in canine kidney cells (MDCK) by our laboratory and the TCID 50 Aliquots were then frozen in liquid nitrogen. The PR8 virus was used fresh each time after aliquoting to avoid repeated freezing and thawing. Staphylococcus aureus (S. aureus) was purchased from the American Type Culture Collection (ATCC).

[0076] 3 Experimental methods

[0077] After one week of adaptive feeding, C57BL / 6J mice (7 weeks of age) were randomly divided into a blank control (NC) group, an influenza virus PR8 infection group, an influenza virus secondary Staphylococcus aureus infection group (PR8+S. aureus), a PR8+OSV treatment group, and a PR8+Jingyin Gubiao Formula (JYGBF) treatment group. The Jingyin Gubiao Formula group received a clinically equivalent dose of 0.233 g / kg, equivalent to a 2.8 g / kg dose for mice; the oseltamivir group received 30 mg / kg daily.

[0078] On the day of infection, the aliquoted influenza virus stock solution was serially diluted with sterile, cold PBS to the experimental concentration and stored on ice until use. Mice were anesthetized using an anesthesia machine. After anesthesia was complete, 50 μL of the diluted virus solution was pipetted dropwise through one nostril. The virus entered the lungs through the mouse's breath, allowing the influenza virus to infect the mouse. After successful infection, the mice were weighed and dosed within 2 hours. Each group was dosed and body weight and symptoms recorded daily for 8 consecutive days. On day 5 of influenza infection, the appropriate titer of Staphylococcus aureus was instilled through the nostrils to establish an influenza virus-induced S. aureus infection model. From day 9 to day 19, mice were weighed but no medication was administered. A weight loss exceeding 30% was defined as a humane endpoint, and mice were sacrificed. Line graphs of weight changes and survival curves were plotted, and statistical differences between groups were calculated. The symptoms of mice were mainly observed to see whether they had "hair ruffling", "arched back posture", "reduced movement", "difficulty breathing", "movement only when stimulated", "no movement when stimulated", "partial closure of eyes", etc. The presence of any of the above symptoms was scored as 1 point.

[0079] 4. Statistics and Analysis

[0080] Same as Example 12.

[0081] 5 Results

[0082] See the results Figure 3 , Figure 3 A is the continuous administration of drugs for 8 days after infection, and the symptom scores of mice were observed and recorded from day 6 to day 9. Figure 1 A shows that the PR8+S. aureus group had more obvious disease symptoms than the blank group. The PR8+OSV treatment group and the PR8+JYGBF treatment group could significantly improve the symptoms of mice secondary infected with influenza and were statistically significant (*P<0.05); Figure 3 B: After infection, the drugs were given for 8 consecutive days, and the survival of the mice was observed and recorded for 19 days. Figure 3 As shown in Figure B, the survival rate of mice in the PR8+S. aureus group was 10%, the survival rate in the OSV group was 100%, and the survival rate in the Jingyin Gubiao formula group was approximately 60%. Compared with the PR8+S. aureus infection model group, the Jingyin Gubiao formula significantly improved the survival rate of mice secondary to influenza infection (*P < 0.05). These results demonstrate that the Jingyin Gubiao formula alleviates symptoms of secondary influenza infection in mice and protects against secondary influenza infection.

[0083] Example 15: The high and low dose groups of Jingyin Gubiao prescription protect mice from weight loss and reduce the lung index of mice secondary to influenza infection

[0084] 1. Drug preparation

[0085] Jingyin Gubiao Fang is Example 1 of the present invention. Jingyin Gubiao Fang granules are prepared and provided by Shanghai Wanshicheng Chinese Medicine Products Co., Ltd. The daily clinical dose of the granules is 14g / person / day (adults). The drug dosage is converted according to the kilogram body weight of humans and mice. The weight of an adult is 60kg. The clinical dosage for humans is 0.233g / kg, and the dosage for mice is 12 times the human dosage, that is, 2.8g / kg. The drug concentration for oral gavage to mice is 0.28g / mL.

[0086] The drug concentration of the low-dose Jingyin Gubiao formula (JYGBF(L)) is 0.28 g / mL, and the high-dose Jingyin Gubiao formula (JYGBF(H)) is 0.56 g / mL. One capsule of oseltamivir (OSV) contains 75 mg, which is dissolved in 25 mL of mineral water for a final concentration of 3 mg / mL.

[0087] 2 Experimental animals and virus strains

[0088] Female, SPF-qualified C57BL / 6J mice, 6 weeks old, weighing 17–20 g, were purchased from the joint venture, SIP-BK Laboratory Animal Co., Ltd. (Shanghai, China). They were housed in a constant temperature (23 ± 2°C), relative humidity (60%–65%), and automatic light–dark cycle (LD 12:12, i.e., 7:00–19:00 light, 19:00–7:00 dark). The animals had free access to food, water, and movement. Mice were acclimated for one week before the experiments. All experiments were conducted in a biosafety level 2 (BSL-2) laboratory approved by the Animal Welfare and Ethics Committee of the Institut Pasteur of the Chinese Academy of Sciences in Shanghai, with the experimental number A2021-L027.

[0089] The mouse-adapted influenza virus strain (influenzavirus A / Puerto Rico / 08 / 1934, PR8) was kindly provided by Professor Xu Ke of the Shanghai Pasteur Institute, Chinese Academy of Sciences. It was amplified in MDCK cells in our laboratory and stored in liquid nitrogen. Staphylococcus aureus (S. aureus) was purchased from the American Type Culture Collection (ATCC).

[0090] 3 Experimental methods

[0091] After one week of adaptive feeding, C57BL / 6J mice (7 weeks of age) were randomly divided into a blank control (NC) group, an influenza virus-secondary Staphylococcus aureus infection group (PR8+S. aureus), a PR8+OSV treatment group, a PR8+Jingyin Gubiao Formula low-dose treatment group [JYGBF(L)], and a PR8+Jingyin Gubiao Formula high-dose treatment group [JYGBF(H)]. The Jingyin Gubiao Formula low-dose group received a clinical equivalent dose of 0.233 g / kg, while the high-dose group received twice the clinical equivalent dose of 0.466 g / kg, which is equivalent to 5.6 g / kg for mice. The oseltamivir group was given 30 mg / kg daily.

[0092] On the day of mouse infection, the aliquoted influenza virus stock solution was gradiently diluted with sterile cold PBS to the experimental concentration and stored on an ice-water mixture until use. Mice were anesthetized using an anesthesia machine. After anesthesia was complete, 50 μL of the diluted virus solution was pipetted dropwise through one nostril, entering the lungs with the mouse's breath, allowing the influenza virus to infect the mice. After successful infection, the mice were weighed and dosed within 2 hours for each group. Dosing was completed daily, and body weight was recorded for 6 consecutive days. On the 5th day of influenza virus infection, the corresponding titer of Staphylococcus aureus was instilled through the nostrils to establish an influenza virus-secondary Staphylococcus aureus infection model.

[0093] After successful infection, mice were weighed, administered, and their weights recorded. On day 6, lung tissues were collected, organ weights were measured, and lung index was calculated.

[0094] 4. Statistics and Analysis

[0095] Same as Example 12.

[0096] 5 Results

[0097] See the results Figure 4 , Figure 4 A shows the weight changes of mice on the sixth day of infection after the mice were infected with the drug for 6 consecutive days. As shown in the figure, after the influenza virus was secondary to Staphylococcus aureus infection, the lung weight of mice was significantly reduced (*P<0.05); the high and low dose groups of Jingyin Gubiao Fang and the OSV treatment group could significantly protect the mice from weight loss after secondary influenza infection (*P<0.05); Figure 4 B. After influenza virus infection followed by Staphylococcus aureus infection, the lung index of mice was significantly reduced (*P < 0.05), suggesting that influenza virus infection may cause pathological damage to the lungs, such as pulmonary edema and hemorrhage. In the OSV-treated group, both the high- and low-dose Jingyin Gubiao formula groups significantly reduced the lung index of influenza-treated mice (*P < 0.05). These results demonstrate that the high- and low-dose Jingyin Gubiao formula of the present invention can protect mice with influenza-induced Staphylococcus aureus infection from weight loss and reduce the lung index.

[0098] Example 16 Protective Effects of High and Low Dose Groups of Jingyin Gubiao Recipe on Lung and Liver Damage Caused by Secondary Influenza Infection

[0099] 1. Drug preparation

[0100] Same as Example 15.

[0101] 2 Experimental animals and virus strains

[0102] Same as Example 15.

[0103] 3 Experimental methods

[0104] After one week of adaptive feeding, C57BL / 6J mice (7 weeks of age) were randomly divided into a blank control (NC) group, an influenza virus-secondary Staphylococcus aureus infection group (PR8+S. aureus), a PR8+OSV treatment group, a PR8+Jingyin Gubiao Fang low-dose treatment group [JYGBF(L)], and a PR8+Jingyin Gubiao Fang high-dose treatment group [JYGBF(H)]. The Jingyin Gubiao Fang low-dose group received a clinical equivalent dose of 0.233 g / kg, while the high-dose group received twice the clinical equivalent dose of 0.466 g / kg. The mice dose should be 2.8 g / kg, and twice the clinical dose is 5.6 g / kg. The oseltamivir group was given 30 mg / kg daily.

[0105] On the day of mouse infection, the aliquoted influenza virus stock solution was gradiently diluted with sterile cold PBS to the experimental concentration and stored on an ice-water mixture until use. Mice were anesthetized using an anesthesia machine. After anesthesia was complete, 50 μL of the diluted virus solution was pipetted dropwise through one nostril, entering the lungs with the mouse's breath, allowing the influenza virus to infect the mice. After successful infection, the mice were weighed and dosed within 2 hours for each group. Dosing was completed daily, and body weight was recorded for 6 consecutive days. On the 5th day of influenza virus infection, the corresponding titer of Staphylococcus aureus was instilled through the nostrils to establish an influenza virus-secondary Staphylococcus aureus infection model.

[0106] After successful infection, the mice were weighed, and the lung and liver tissues of the mice were obtained on the 6th day of infection and stained with hematoxylin-eosin (HE) to observe the protective effect of JYGBF on the lungs and livers of mice secondary to influenza infection.

[0107] 4 Results

[0108] See the results Figure 5As shown in the figure, after secondary influenza infection, the lung tissue of infected mice showed massive hemorrhage, consolidation and edema, a large number of white granulocytes entered the bronchiolar lumen, the pulmonary interstitium widened, and was characterized by multifocal lymphocyte infiltration. The liver sinusoidal structure of the mice was destroyed, the arrangement of the liver lobules was disordered, and the infiltration of inflammatory cells increased. In OSV, after treatment with the high and low dose groups of Jingyin Gubiao Fang, the alveolar tissue of the mice was protected, the infiltration of inflammatory cells was significantly reduced, and the liver structure remained stable. The results show that the high and low dose groups of Jingyin Gubiao Fang have a protective effect on the damage to the lungs and livers of mice secondary to influenza infection.

[0109] Example 17: The high and low dose groups of Jingyin Gubiao prescription reduce the protein expression levels of inflammatory factors in the lungs of mice with secondary influenza infection

[0110] 1. Drug preparation

[0111] Same as Example 15.

[0112] 2 Experimental animals and virus strains

[0113] Same as Example 15.

[0114] 3 Experimental methods

[0115] After one week of adaptive feeding, C57BL / 6J mice (7 weeks of age) were randomly divided into a blank control (NC) group, an influenza virus-secondary Staphylococcus aureus infection group (PR8+S. aureus), a PR8+OSV treatment group, a PR8+Jingyin Gubiao Fang low-dose treatment group [JYGBF(L)], and a PR8+Jingyin Gubiao Fang high-dose treatment group [JYGBF(H)]. The Jingyin Gubiao Fang low-dose group received a clinical equivalent dose of 0.233 g / kg, while the high-dose group received twice the clinical equivalent dose of 0.466 g / kg. The mice dose should be 2.8 g / kg, and twice the clinical dose is 5.6 g / kg. The oseltamivir group was given 30 mg / kg daily.

[0116] On the day of mouse infection, the aliquoted influenza virus stock solution was gradiently diluted with sterile cold PBS to the experimental concentration and stored on an ice-water mixture until use. Mice were anesthetized using an anesthesia machine. After anesthesia was complete, 50 μL of the diluted virus solution was pipetted dropwise through one nostril, entering the lungs with the mouse's breath, allowing the influenza virus to infect the mice. After successful infection, the mice were weighed and dosed within 2 hours for each group. Dosing was completed daily, and body weight was recorded for 6 consecutive days. On the 5th day of influenza virus infection, the corresponding titer of Staphylococcus aureus was instilled through the nostrils to establish an influenza virus-secondary Staphylococcus aureus infection model.

[0117] After successful infection, mice were weighed, administered, and their weights recorded. On day 6 of infection, lung tissue was harvested and homogenates were prepared. Cytokine analysis of the homogenate supernatant was performed using ELISA.

[0118] 4. Statistics and Analysis

[0119] Same as Example 12.

[0120] 5 Results

[0121] See the results Figure 6 , Figure 6 AF represents the protein expression of the lung inflammatory factors TNF-α, IL-6, CCL5, MIP-2, ICAM1, and IL-10 on day 6 of infection, following six consecutive days of administration. As shown in the figure, both the high- and low-dose groups of Jingyin Gubiao Formula reduced the expression of the lung cytokines TNF-α, IL-6, CCL5, MIP-2, and ICAM1 (*P < 0.05); the JYGBF(H) treatment group reduced the expression of the lung cytokine IL-10. These results indicate that both the high- and low-dose groups of Jingyin Gubiao Formula can reduce the expression of lung cytokine proteins in mice secondary to influenza infection.

[0122] Example 18: The high and low dose groups of Jingyin Gubiao prescription reduce the mRNA expression levels of inflammatory factors in the lungs of mice with secondary influenza infection

[0123] 1. Drug preparation

[0124] Same as Example 15.

[0125] 2 Experimental animals and virus strains

[0126] Same as Example 15.

[0127] 3 Experimental methods

[0128] After one week of adaptive feeding, C57BL / 6J mice (7 weeks of age) were randomly divided into a blank control (NC) group, an influenza virus-secondary Staphylococcus aureus infection group (PR8+S. aureus), a PR8+OSV treatment group, a PR8+Jingyin Gubiao Fang low-dose treatment group [JYGBF(L)], and a PR8+Jingyin Gubiao Fang high-dose treatment group [JYGBF(H)]. The Jingyin Gubiao Fang low-dose group received a clinical equivalent dose of 0.233 g / kg, while the high-dose group received twice the clinical equivalent dose of 0.466 g / kg. The mice dose should be 2.8 g / kg, and twice the clinical dose is 5.6 g / kg. The oseltamivir group was given 30 mg / kg daily.

[0129] On the day of mouse infection, the aliquoted influenza virus stock solution was gradiently diluted with sterile cold PBS to the experimental concentration and stored on an ice-water mixture until use. Mice were anesthetized using an anesthesia machine. After anesthesia was complete, 50 μL of the diluted virus solution was pipetted dropwise through one nostril, entering the lungs with the mouse's breath, allowing the influenza virus to infect the mice. After successful infection, the mice were weighed and dosed within 2 hours for each group. Dosing was completed daily, and body weight was recorded for 6 consecutive days. On the 5th day of influenza virus infection, the corresponding titer of Staphylococcus aureus was instilled through the nostrils to establish an influenza virus-secondary Staphylococcus aureus infection model.

[0130] After successful infection, mice were weighed, administered, and their weights recorded. On day 6 of infection, lung tissue was harvested and homogenized, and total lung RNA was extracted. Lung cytokine mRNA expression was analyzed using QPCR.

[0131] 4. Statistics and Analysis

[0132] Same as Example 12.

[0133] 5 Results

[0134] See the results Figure 7 , Figure 7 AD represents the mRNA expression of lung inflammatory factors TNF-α, CCL5, MIP-2, and MCP-1 on the sixth day of infection after 6 consecutive days of administration. As shown in the figure, different doses of JYGBF treatment groups can reduce the expression of lung cytokines TNF-α, CCL5, and MIP-2 (*P<0.05); the JYGBF (H) treatment group can reduce the expression of lung cytokine MCP-1. These results show that the high and low dose groups of the Jingyin Gubiao prescription of the present invention can reduce the mRNA expression of lung cytokines in mice secondary to influenza infection.

[0135] Example 19: The high and low dose groups of Jingyin Gubiao prescription reduce the expression levels of serum inflammatory factor proteins in mice secondary to influenza infection

[0136] 1. Drug preparation

[0137] Same as Example 15.

[0138] 2 Experimental animals and virus strains

[0139] Same as Example 15.

[0140] 3 Experimental methods

[0141] After one week of adaptive feeding, C57BL / 6J mice (7 weeks of age) were randomly divided into a blank control (NC) group, an influenza virus-secondary Staphylococcus aureus infection group (PR8+S. aureus), a PR8+OSV treatment group, a PR8+Jingyin Gubiao Fang low-dose treatment group [JYGBF(L)], and a PR8+Jingyin Gubiao Fang high-dose treatment group [JYGBF(H)]. The Jingyin Gubiao Fang low-dose group received a clinical equivalent dose of 0.233 g / kg, while the high-dose group received twice the clinical equivalent dose, 0.466 g / kg. The dose for mice should be 2.8 g / kg; twice the clinical dose for mice is 5.6 g / kg. The oseltamivir group was given 30 mg / kg daily.

[0142] On the day of mouse infection, the aliquoted influenza virus stock solution was gradiently diluted with sterile cold PBS to the experimental concentration and stored on an ice-water mixture until use. Mice were anesthetized using an anesthesia machine. After anesthesia was complete, 50 μL of the diluted virus solution was pipetted dropwise through one nostril, entering the lungs with the mouse's breath, allowing the influenza virus to infect the mice. After successful infection, the mice were weighed and dosed within 2 hours for each group. Dosing was completed daily, and body weight was recorded for 6 consecutive days. On the 5th day of influenza virus infection, the corresponding titer of Staphylococcus aureus was instilled through the nostrils to establish an influenza virus-secondary Staphylococcus aureus infection model.

[0143] After successful infection, mice were weighed, administered, and their weights recorded. On day 6 of infection, serum was obtained from mice and cytokine protein expression in the serum was analyzed using CBA technology.

[0144] 4. Statistics and Analysis

[0145] Same as Example 12.

[0146] 5 Results

[0147] See the results Figure 8 , Figure 8 AC represents the protein expression of serum inflammatory factors IL-6, TNF-α, and IFN-γ on the sixth day of infection after continuous administration for 6 days. As shown in the figure, different doses of JYGBF treatment groups can reduce the protein expression of lung cytokines IL-6, TNF-α, and IFN-γ (*P<0.05). These results indicate that the high- and low-dose groups of the Jingyin Gubiao prescription of the present invention can reduce the expression of cytokine proteins in the serum of mice secondary to influenza infection.

[0148] In summary, the Jingyin Gubiao prescription provided by the present invention has a good in vivo protective effect on mice secondary to influenza infection, significantly alleviating inflammatory reactions and organ damage. Furthermore, the nine Chinese herbs in the composition of the present invention, namely honeysuckle, astragalus, siler, patchouli, isatis root, schizonepeta, reed root, platycodon, and liquorice, all have the advantages of being widely available, safe, low-toxic, and low-cost, making them very suitable for large-scale promotion and application, with significant social benefits and economic value.

[0149] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a traditional Chinese medicine composition for treating influenza and influenza-related bacterial infections in the preparation of a medicament for treating influenza-related bacterial infections, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 3-15 parts of honeysuckle, 3-15 parts of schizonepeta, 6-18 parts of astragalus, 3-15 parts of patchouli, 3-15 parts of siler, 3-15 parts of isatis root, 3-15 parts of platycodon, 9-21 parts of reed rhizome, and 2-8 parts of liquorice.

2. The use according to claim 1, characterized in that The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 6-12 parts of honeysuckle, 6-12 parts of schizonepeta, 9-15 parts of astragalus, 6-12 parts of patchouli, 6-12 parts of siler, 6-12 parts of isatis root, 6-12 parts of platycodon, 12-18 parts of reed rhizome, and 4-8 parts of liquorice.

3. The use according to claim 1, characterized in that The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 9 parts of honeysuckle, 9 parts of schizonepeta, 12 parts of astragalus, 9 parts of patchouli, 9 parts of siler, 9 parts of isatis root, 9 parts of platycodon, 15 parts of reed rhizome and 6 parts of liquorice.

Images