A traditional Chinese medicine composition for treating post-infectious cough, and its traditional Chinese medicine preparation, preparation method and application

Abstract

The present invention discloses a traditional Chinese medicine composition for treating post-infectious cough, as well as its preparation, preparation method, and application, belonging to the field of post-infectious cough medicine. The traditional Chinese medicine composition for treating post-infectious cough proposed by the present invention comprises the following raw materials: mulberry leaf, roasted ephedra, golden fecundation, honey white qian, perilla seed, loquat leaf, burdock fruit, thunbergia bulb, scrophularia, bombyx batryticatus, platycodon grandiflorum, and roasted liquorice root, with a mass ratio of 5-15:6-10:5-15:5-15:5-15:5-15:5-15:5-15:5-15:5-15:5-15:3-9. The traditional Chinese medicine composition proposed by the present invention highly conforms to the pathogenesis of PIC, which is "wind evil invading the lungs, lung obstruction and qi reversal." It is a clinically effective prescription for treating chronic and subacute coughs such as PIC. It has good therapeutic effects on both wind-cold cough and wind-heat cough, cough with or without phlegm, and can effectively reduce cough severity and cough sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of medicines for treating post-infectious cough, and particularly relates to a traditional Chinese medicine composition for treating post-infectious cough, a traditional Chinese medicine preparation thereof, a preparation method and an application thereof. Background Art

[0002] Post-infectious cough (PIC) is a persistent cough that persists after an upper respiratory tract infection (URI) despite resolution of acute symptoms and normal chest X-ray findings. Clinically, it presents with an irritating, dry cough and pharyngeal discomfort, or with a small amount of white mucus, lasting 3 to 8 weeks. It is typically triggered by viruses, bacteria, or mycoplasmas. Its pathogenesis is complex, involving a multifactorial interaction. Airway neurogenic inflammation and airway hyperresponsiveness are the core mechanisms of PIC. Cytokines released by immune cells activate TRPV1, increasing the sensitivity of peripheral nerve endings. Overactivation and sensitization of airway sensory nerves lead to potential tissue damage and cough hypersensitivity. After respiratory infection, pathogens activate immune cells, releasing large amounts of cytokines that damage the airway mucosal epithelium, disrupting airway barrier function and increasing airway mucosal permeability, making cough more susceptible to external stimuli such as cold air. Furthermore, an imbalance in the secretion of inflammatory cytokines after infection contributes to persistent airway neurogenic inflammation and airway hyperresponsiveness throughout PIC, ultimately causing recurrent cough. COX-2, PGE-2, and TXA-2 are cough-causing factors closely associated with airway inflammation and are significantly elevated during PIC. The inflammation-inducible cyclooxygenase COX-2 promotes arachidonic acid metabolism, producing PGE-2 and TXA-2, thereby exacerbating the inflammatory response. PGE-2 can directly stimulate unmyelinated sensory C fibers in the lungs, inducing cough. It can also lower the activation threshold of transient receptor potential vanilloid type 1 (TRPV1) channels, enhancing the cough reflex induced by irritants such as capsaicin, thus contributing to the development of PIC. TXA-2 binds to TP receptors on airway smooth muscle cells, triggering a sudden increase in intracellular calcium ion concentration, leading to strong bronchoconstriction and significantly increasing the sensitivity of airway sensory nerves to irritants such as capsaicin and bradykinin. TXA-2 can also promote ROS expression through a NOX-dependent pathway, exacerbating inflammation.

[0003] PIC is classified as a chronic or stubborn cough in Traditional Chinese Medicine. Its core pathogenesis is caused by wind pathogens invading the lungs, causing wind-induced qi reversal, or by the presence of phlegm and heat pathogens. The Sangma Zhike Formula (SMZKF) is a clinically effective prescription for the treatment of PIC, demonstrating its efficacy in dispelling wind pathogens, promoting lung function, relieving sore throat, and alleviating cough. However, further research is needed. TRPV1 activation, mediating the release of substance P (SP) and calcitonin gene-related peptide (CGRP) from sensory nerve endings (such as C fibers), and the pyroptosis pathway are the core regulatory mechanisms of cough sensitivity and airway inflammation in PIC, respectively. Currently, there is no specific treatment for PIC. Symptomatic treatment with antitussive, antihistamine, anti-inflammatory, and decongestant medications is common, but these medications are prone to side effects such as drowsiness, dry mouth, and loss of appetite, and relapse is common after discontinuation of these medications. Therefore, the development of safe, effective, and well-defined therapeutic agents is of great clinical significance. Summary of the Invention

[0004] In response to the above problems, in the first aspect, the present invention proposes a traditional Chinese medicine composition for treating post-infectious cough, comprising the following raw materials: mulberry leaves, roasted ephedra, golden fecundation, honey whitehead, perilla seeds, loquat leaves, burdock seeds, thunbergia bulb, Scrophularia ningpoensis, bombyx batryticatus, platycodon and roasted licorice, with a mass ratio of 5-15:6-10:5-15:5-15:5-15:5-15:5-15:5-15:5-15:5-15:5-15:3-9.

[0005] Furthermore, the Chinese medicine composition includes the following raw materials: mulberry leaves, roasted ephedra, golden fecundation, honey whitehead, perilla seeds, loquat leaves, burdock seeds, thunbergia bulb, Scrophularia ningpoensis, bombyx batryticatus, platycodon and roasted licorice, with a mass ratio of 8-12:7-9:8-12:8-12:8-12:8-12:8-12:8-12:8-12:8-12:8-12:4-7.

[0006] Furthermore, the traditional Chinese medicine composition includes the following raw materials: 5-15g of mulberry leaves, 6-10g of roasted ephedra, 5-15g of jinfeicao, 5-15g of honey baiqian, 5-15g of perilla seeds, 5-15g of loquat leaves, 5-15g of burdock seeds, 5-15g of thunbergia bulb, 5-15g of Scrophularia ningpoensis, 5-15g of bombyx batryticatus, 5-15g of platycodon and 3-9g of roasted licorice.

[0007] Furthermore, the traditional Chinese medicine composition includes the following raw materials: 8-12g of mulberry leaves, 7-9g of roasted ephedra, 8-12g of jinfeicao, 8-12g of honey baiqian, 8-12g of perilla seeds, 8-12g of loquat leaves, 8-12g of burdock seeds, 8-12g of thunbergia bulb, 8-12g of Scrophularia ningpoensis, 8-12g of bombyx batryticatus, 8-12g of platycodon and 4-7g of roasted licorice.

[0008] Furthermore, the traditional Chinese medicine composition includes the following raw materials: 10g of mulberry leaves, 8g of roasted ephedra, 10g of jinfeicao, 10g of honey baiqian, 10g of perilla seeds, 10g of loquat leaves, 10g of burdock seeds, 10g of thunbergia bulb, 10g of Scrophularia ningpoensis, 10g of bombyx batryticatus, 10g of platycodon and 6g of roasted licorice.

[0009] In a second aspect, the present invention provides a method for preparing a Chinese medicine composition, comprising the following steps:

[0010] Soaking various raw materials in the traditional Chinese medicine composition in water for 80-100 minutes, boiling and then decocting for 60-120 minutes, filtering to obtain a decoction and a residue;

[0011] The residue is soaked in water, boiled and then decocted for 30-50 minutes, filtered to obtain a decoction, and the two decoctions are combined and concentrated to obtain a traditional Chinese medicine composition. Specifically, the concentration of the traditional Chinese medicine composition is 0.50325 g / mL-2.013 g / mL.

[0012] Furthermore, the mass-to-volume ratio of the traditional Chinese medicine formula to distilled water is 1:5-20 g / L.

[0013] Furthermore, the mass volume ratio of the residue to distilled water is 0.5-1.5:8 g / L.

[0014] In a third aspect, the present invention provides a traditional Chinese medicine preparation, which contains the traditional Chinese medicine composition.

[0015] Furthermore, the preparation types of the traditional Chinese medicine preparation include tablets, capsules, granules, pills, decoctions or powders.

[0016] In a fourth aspect, the present invention proposes the use of the traditional Chinese medicine composition in the preparation of a medicine for treating post-infectious cough.

[0017] Furthermore, the Chinese medicine composition improves post-infectious cough by simultaneously acting on the TRPV1-mediated SP / NK1R and CGRP release, as well as the cell pyroptosis pathway.

[0018] Beneficial effects of the present invention:

[0019] The traditional Chinese medicine composition proposed in this paper is highly consistent with the pathogenesis of PIC, characterized by "wind evil invading the lungs, causing obstruction of lung qi," and is a clinically proven formula for treating chronic and subacute coughs such as PIC. It demonstrates excellent therapeutic efficacy for treating both wind-cold and wind-heat coughs, and coughs with or without phlegm, effectively reducing cough severity and sensitivity in PIC patients. Mulberry leaves in this formula dispel wind and pathogenic factors, clearing latent heat in the lungs and moistening the lungs. Roasted ephedra, with its mild warming and drying properties, specifically dispels pathogenic factors, promotes lung qi, and relieves cough. Combined with mulberry leaves, they offer a cooling and warming effect, dispersing pathogenic factors without contributing to heat, and moistening without astringing pathogenic factors. Together, these two herbs serve as the main ingredients. Burdock seeds dispel wind and pathogenic factors, open up the lungs and relieve sore throats, and help mulberry leaves and roasted ephedra to expel pathogenic factors; silkworm pupa dispels internal and external wind, resolves phlegm and disperses stagnation, and relieves airway spasm; golden fecundation descends qi and eliminates phlegm, dredges the lungs, and serves as a minister; honey white plum descends qi, eliminates phlegm, and stops coughs caused by lung qi stagnation; loquat leaves clear the lungs and descend counterflow, resolve phlegm and relieve coughs, and directly relieve lung heat; perilla seeds descend qi and eliminate phlegm, and treat coughs caused by counterflow of qi, and are used for coughs and asthma caused by phlegm stagnation; thungbei clears heat and disperses stagnation, resolves phlegm and relieves coughs, and is used for stubborn coughs caused by phlegm stagnation and qi reversal; scrophularia nourishes yin, reduces fire and relieves sore throats, and prevents phlegm heat or lung heat from damaging yin; platycodon opens the lungs and relieves sore throats, carries medicine upward, opens and lifts lung qi to help expel phlegm, and is used together with other medicines that descend lung qi, one to open and one to descend, to restore the function of lung qi, and serves as an adjuvant; roasted licorice moistens the lungs and relieves coughs, and harmonizes other medicines as a guiding medicine. All these medicines work together to dispel wind and evil, promote lung function, relieve throat and cough.

[0020] The traditional Chinese medicine composition proposed in the present invention can effectively improve cough sensitivity and airway neurogenic inflammation in PIC model rats. Its mechanism of action is closely related to the inhibition of TRPV1-mediated SP / NK1R and CGRP release and cell pyroptosis pathway, laying a solid foundation for the development of new anti-PIC drugs and having significant clinical application value and market prospects.

[0021] Other features and advantages of the present invention will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 A schematic diagram of the construction and drug administration of a PIC animal model in an embodiment of the present invention is shown;

[0024] Figure 2AThe figures show the statistical results of the cough latency of rats in each group in the embodiment of the present invention;

[0025] Figure 2B The statistical results of the number of coughs in 5 minutes in each group of rats in the embodiment of the present invention are shown;

[0026] Figure 3A The figure shows the pathological results of HE staining of lung tissues of rats in each group in the embodiment of the present invention (including alveoli and bronchi);

[0027] Figure 3B The figure shows the scoring results of bronchial inflammation in rats in each group according to the embodiment of the present invention;

[0028] Figure 3C The figure shows the scoring results of alveolar inflammation in rats in each group according to the embodiment of the present invention;

[0029] Figure 4A The white blood cell (WBC) count results in the bronchoalveolar lavage fluid (BALF) of each group of rats in the embodiment of the present invention are shown;

[0030] Figure 4B The figures show the lymphocyte (LYM) count results in BALF of rats in each group in the embodiment of the invention;

[0031] Figure 4C The figures show the results of neutrophil (NEU) counting in BALF of rats in each group in the embodiment of the invention;

[0032] Figure 4D The figures show the counting results of mononuclear macrophages (MONs) in BALF of rats in each group in the examples of the invention;

[0033] Figure 4E The figures show the results of eosinophil counts (EOS) in BALF of rats in each group in the examples of the invention;

[0034] Figure 5A The expression of IL-1β in BALF of rats in each group in the embodiment of the present invention is shown;

[0035] Figure 5B The expression of TNF-α in BALF of rats in each group according to the embodiment of the present invention is shown;

[0036] Figure 5C The expression of IL-6 in BALF of rats in each group in the embodiment of the present invention is shown;

[0037] Figure 5D The expression of COX-2 in BALF of rats in each group in the embodiment of the present invention is shown;

[0038] Figure 5EThe expression of PGE2 in BALF of rats in each group in the embodiment of the present invention is shown;

[0039] Figure 5F The expression of TXA2 in BALF of rats in each group according to the embodiment of the present invention is shown;

[0040] Figure 6A The expression of ROS in the lung tissue of each group of rats in the embodiment of the present invention is shown;

[0041] Figure 6B The expression of MDA in the lung tissue of each group of rats in the embodiment of the present invention is shown;

[0042] Figure 6C The expression of MPO in the lung tissue of each group of rats in the embodiment of the present invention is shown;

[0043] Figure 7A The Western blot images of TRPV1, SP, CGRP and NK1R in the lung tissues of rats in each group according to the embodiment of the present invention are shown, with GAPDH as the internal reference protein;

[0044] Figure 7B Statistical graphs showing the expression of TRPV1 in lung tissue of rats in each group according to the embodiment of the present invention are shown;

[0045] Figure 7C Statistical graphs showing the expression of SP in lung tissue of rats in each group according to the embodiment of the present invention are shown;

[0046] Figure 7D The figure shows the statistical graph of CGRP expression in the lung tissue of each group of rats in the embodiment of the present invention;

[0047] Figure 7E Statistical graphs showing the expression of NK1R in lung tissue of rats in each group according to the embodiment of the present invention are shown;

[0048] Figure 8A Western blots of P-NF-κB, NLRP3, ACS, Cleaved-Caspase-1, Cleaved-IL-1β, and GSDMD-N in the lung tissues of rats in each group according to the present invention are shown, with GAPDH as an internal reference protein;

[0049] Figure 8B The figure shows the statistical graph of the expression of P-NF-κB in the lung tissue of each group of rats in the embodiment of the present invention;

[0050] Figure 8C Statistical graphs showing the expression levels of NLRP3 in lung tissues of rats in each group according to the embodiment of the present invention are shown;

[0051] Figure 8DThe statistical graph of ACS expression in lung tissue of each group of rats in the embodiment of the present invention is shown;

[0052] Figure 8E The statistical graph of the expression of Cleaved-Caspase-1 in the lung tissue of each group of rats in the embodiment of the present invention is shown;

[0053] Figure 8F The statistical graph of the expression of Cleaved-IL-1β in the lung tissue of each group of rats in the embodiment of the present invention is shown;

[0054] Figure 8G Statistical graphs of the expression levels of GSDMD-N in the lung tissues of rats in each group in the examples of the present invention are shown. DETAILED DESCRIPTION

[0055] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0056] Some of the raw materials, reagents and instruments involved in the following examples are as follows:

[0057] Lipopolysaccharide (LPS) (SIGMA, 93572-42-0), hematoxylin stain (ebiogo, B006), alcohol-soluble eosin stain (ebiogo, B005), rat interleukin-1β (IL-1β) ELISA kit (Wuhan Gene Beauty, JYM0419Ra), rat tumor necrosis factor α (TNF-α) ELISA kit (Wuhan Gene Beauty, JYM0635Ra), rat IL-6 ELISA kit (Wuhan Gene Beauty, JYM0646Ra), rat cyclooxygenase-2 (COX-2) kit (Wuhan Gene Beauty, JYM0885Ra), rat prostaglandin E2 (Prostaglandin E2) ELISA kit (Wuhan Gene Beauty, JYM0885Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM0890Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM0910Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM0920Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM0930Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM0940Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM0950Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM0960Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM0970Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM0980Ra), rat cytokine release assay (Wuhan Gene Beauty, JYM09 ... E2, PGE2) kit (Wuhan Gene Beauty, JYM0446Ra), rat thromboxane A2 (TXA2) kit (Wuhan Gene Beauty, JYM0893Ra), malondialdehyde (MDA) test kit (Nanjing Jiancheng, A003-1), myeloperoxidase (MPO) test kit (Nanjing Jiancheng, A044-1-1), reactive oxygen species (ROS) test kit (Nanjing Jiancheng, A044-1-1), Species, ROS) detection kit (Biyuntian, S0033S), PVDF membrane (Millipore, IPVH00010), PBS buffer powder (Zs-BIO, ZLI-9062), prestained protein marker (Thermo, 26617), western rapid transfer buffer (Beyotime, P0575-10L), ECL ultrasensitive luminescence kit (Biosharp, BL520A), goat anti-mouse IgG (Zsbio, ZB-2305), goat anti-rabbit IgG (Zsbio, ZB-2301), phosphorylated nuclear factor kappa-B (P-NF-κB) (ImmunoWay.Biotechnology, YP0191), NOD-like receptor pyrin domain-associated protein 3 (NLR Family Pyrin Domain Containing 3, NLRP3) (Abcam, ab263899), Apoptosis-Associated Speck-like Protein Containing a CARD (ASC) (Affinity, DF6304), Cleaved Cysteinyl Aspartate Specific Proteinase-1 (Cleaved-Caspase-1) (Affinity, AF4005), Gasdermin D N-terminal Domain (gSDMD-N) (Santa Cruz, sc-393581), and Cleaved-IL-1β (Wanleibio, WL00891). SP (Abcam, ab239503), TRPV1 (Bioworld, BS60454), Neurokinin-1 Receptor (NK1R) (Bioss, bs-0064R), CGRP (Santa Cruz, sc-57053), GAPDH (Zsbio, TA-08).

[0058] Microplate reader (Raydu, RT-6000), UV-visible spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd., UV-1800), centrifuge (Anhui Jiawen, JW3021HR), vortex mixer (Qilin Bell Instrument Manufacturing Co., Ltd., GL-88B), electric heating constant temperature box (Shanghai Sanfa, DNP-9052BS-Ⅲ), -80℃ refrigerator (Panasonic, MDF-U33865), electrophoresis apparatus (Tanon, EPS300), electrophoresis tank (Tanon, VE-180), membrane transfer apparatus (Tanon, VE-186), automatic exposure instrument (Shanghai Peiqing Technology Co., Ltd., JS-1070P), cell counter (Countstar, IC 1000).

[0059] Example 1

[0060] This example exemplifies the specific composition of a traditional Chinese medicine composition (abbreviated as SMZKF) for treating post-infectious cough. All the medicinal materials involved were purchased from the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine.

[0061] SMZKF 1: mulberry leaves 10g, roasted ephedra 8g, golden feicui 10g, honey baiqian 10g, perilla seeds 10g, loquat leaves 10g, burdock seeds 10g, thunbergia 10g, Scrophularia 10g, Bombyx batryticatus 10g, Platycodon 10g and roasted liquorice 6g.

[0062] SMZKF II: mulberry leaves 5g, roasted ephedra 6g, golden feicui 5g, honey baiqian 5g, perilla seeds 5g, loquat leaves 5g, burdock seeds 5g, thunbergia thunbergii 5g, Scrophularia 5g, bombyx batryticatus 5g, platycodon 5g and roasted liquorice 3g.

[0063] SMZKF Three: mulberry leaves 15g, roasted ephedra 10g, golden feicui 15g, honey baiqian 15g, perilla seeds 15g, loquat leaves 15g, burdock seeds 15g, thunbergia bulb 15g, Scrophularia 15g, bombyx batryticatus 15g, platycodon 15g and roasted liquorice 9g.

[0064] SMZKF Four: mulberry leaves 15g, roasted ephedra 10g, golden feicui 10g, honey baiqian 10g, perilla seeds 10g, loquat leaves 10g, burdock seeds 10g, thunbergia 10g, Scrophularia 10g, Bombyx batryticatus 10g, Platycodon 10g and roasted liquorice 9g.

[0065] SMZKF Five: mulberry leaves 10g, roasted ephedra 8g, golden feicui 6g, honey baiqian 5g, perilla seeds 5g, loquat leaves 5g, burdock seeds 5g, thunbergia 5g, Scrophularia 5g, bombyx batryticatus 5g, platycodon 5g and roasted liquorice 3g.

[0066] SMZKF Six: Mulberry leaves 8g, roasted ephedra 8g, golden feicui 8g, honey baiqian 8g, perilla seeds 8g, loquat leaves 8g, burdock seeds 8g, thunbergia bulb 8g, Scrophularia 8g, bombyx batryticatus 8g, platycodon 8g and roasted liquorice 8g.

[0067] SMZKF Seven: mulberry leaves 12g, roasted ephedra 9g, golden feicui 12g, honey baiqian 12g, perilla seeds 12g, loquat leaves 12g, burdock seeds 12g, thunbergia 12g, Scrophularia 12g, bombyx batryticatus 12g, platycodon 12g and roasted liquorice 8g.

[0068] Preparation of the Chinese medicine composition: Soak the medicinal materials in distilled water (mass volume ratio of medicinal materials to distilled water is 1:10 g / L) according to the composition ratio of SMZKF-7 for 60 minutes, boil over high heat, and then simmer over low heat for 90 minutes;

[0069] The decoction was filtered through four layers of gauze, and the residue was soaked in distilled water (the mass volume ratio of the residue to distilled water was 1:8 g / L). After boiling over high heat, it was simmered over low heat for 40 minutes. The mixture was filtered again, and the two decoctions were combined and concentrated. The concentrated product (concentration of 2.013 g / mL, i.e., the traditional Chinese medicine composition) was stored at 4°C for later use.

[0070] The above-mentioned Chinese medicine composition can also be formed into corresponding preparations, and the preparation type can be tablets, capsules, granules, pills, decoctions or powders.

[0071] Control drug: Compound methoxyphenamine capsules (produced by Jiangsu Hanjing Pharmaceutical Co., Ltd., national medicine approval number: H20033669).

[0072] Example 2

[0073] The Chinese medicine composition proposed in Example 1 was subjected to animal efficacy tests.

[0074] (1) Construction of animal model and grouping:

[0075] Modeling and Grouping and Dosing: All SD rats were randomly divided into a control group (CON), a model group (PIC), a low-dose SMZKF group (SMZKF-L), a medium-dose SMZKF group (SMZKF-M), a high-dose SMZKF group (SMZKF-H), and an ASM group (8 rats per group). Except for the CON group, the PIC rat model was established using a combination of smoke fumigation, lipopolysaccharide nasal drops, and capsaicin aerosolization. SMZKF in each dose group was prepared by diluting the concentrated product described in Example 1.

[0076] according to Figure 1 The animal model was constructed and drug was administered. The specific process is as follows:

[0077] On the 1st to 10th day, smoke fumigation was used to establish airway hyperresponsiveness. Rats were placed in a fumigation box and fumigated with 50 g of sawdust and 5 cigarettes for 30 minutes each time, twice a day.

[0078] On days 11, 14, and 17 of the experiment, rats were anesthetized with isoflurane and 0.4 mg / mL LPS solution was instilled into the rats' nasal cavity at a volume of 1 μL / g once daily. On days 12, 13, 15, 16, and 18 of the experiment, rats were placed in a sealed container and a 0.1 mmol / L capsaicin solution was aerosolized and inhaled once daily to induce cough.

[0079] On the 18th day after modeling, the rats were subjected to a capsaicin cough provocation test. If the rats showed characteristic signs such as coughing, accelerated breathing, leg extension, neck extension, abdominal muscle contraction, and coughed more than 10 times within 3 minutes, the model was successfully established.

[0080] On day 19, intragastric administration (IG) was initiated, with doses calculated according to the equivalent dose ratio table for humans and animals based on surface area. The CON and PIC groups received normal saline (10 mL / kg gavage) once daily. SMZKF was administered gavage once daily at a low dose of 5.03 g / kg, a medium dose of 10.06 g / kg, and a high dose of 20.13 g / kg. The ASM group received compound methoxyphenamine capsules gavage (24.63 mg / kg) once daily for 10 days. After 10 days, rats were euthanized and samples of BALF, abdominal aortic serum, and lung tissue were collected for analysis.

[0081] (2) Observe the general condition and behavioral characteristics of rats before and after modeling:

[0082] General behavioral observations: Before modeling, rats in the CON group were in good spirits, with smooth and shiny fur, normal eating, and even breathing. After modeling, rats in all groups became less spirited, with messy and less shiny fur, decreased eating, and accelerated breathing. After capsaicin atomization, rats exhibited obvious coughing, sneezing, face scratching, and accelerated breathing. All groups showed varying degrees of improvement after drug treatment.

[0083] Behavioral observations included observing the cough sensitivity of each rat group after modeling, including cough latency and cough frequency. Specifically, one hour after the last dose, capsaicin aerosol was used to induce coughing for 3 minutes. The rats' cough latency and the number of coughs within 5 minutes were recorded. (Characteristic coughing movements of rats include accelerated breathing, neck and front leg extension, mouth opening, and abdominal contraction.)

[0084] The latency period and frequency of coughing in mice of different groups were compared. Figure 2A and Figure 2B As shown, Figure 2A The statistical data of rat cough latency is shown in Figure 2. Figure 2B The statistical data of the number of coughs are shown in Table 1. Compared with the CON group, the cough latency of rats in the PIC group was significantly shortened, and the number of coughs within 5 minutes was significantly increased. The difference was statistically significant ( P <0.05), indicating that the rats in the PIC group developed high sensitivity to airway cough and the model was successfully established. Compared with the PIC group, the SMZKF-L group, SMZKF-M group, and SMZKF-H group could prolong the cough latency and reduce the number of coughs within 5 minutes, showing a certain dose-dependent manner ( P <0.05), and the effect of high-dose SMZKF was similar to that of compound methoxyphenamine ( P >0.05).

[0085] (3) Pathological observation of rat lung tissue: Lung tissues of rats were collected from the same location, fixed with 4% neutral paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin-eosin (HE), and sealed with neutral gum. Histopathological morphology was then observed and analyzed under a light microscope. Refer to relevant literature for peribronchial inflammation (RidzuanN, ZakariaN, WideraD, et al. Human umbilical cord mesenchymal stem cell-derivedextracellular vesicles ameliorate airway inflammation in a rat model of chronic obstructive pulmonary disease(COPD)[J]. Stem Cell Res Ther. 2021, 12(1): 54.]; Yang Qinjun, Wang Hui, Xu Shuyu, et al. The mechanism of Shenqi Tiaoshen prescription in alleviating airway inflammation in rats with chronic obstructive pulmonary disease and lung and kidney qi deficiency syndrome: based on ferroptosis pathway[J]. Journal of Southern Medical University, 2024, 44(10): 1937-1946.) and alveolar inflammation (Yang Y, Huang L, Tian C, Qian B. Magnesium isoglycyrrhizinate inhibits airway inflammation in rats with chronic obstructive pulmonary disease[J]. BMC PulmMed.2021,21(1):371.) for scoring.

[0086] Peribronchial inflammation was scored as follows: no inflammation, 0 points; occasional inflammatory cell infiltration, 1 point; a thin layer of inflammatory cells (1-5 cells thick) surrounding most bronchi, 2 points; and a thick layer of inflammatory cells (>5 cells thick) surrounding most bronchi and blood vessels, 3 points. Alveolar inflammation was scored as follows: normal cells, 0 points; a small amount of inflammatory cell infiltration, 1 point; an inflammatory cell ring >1 cell layer deep, 2 points; an inflammatory cell ring >2-4 cell layers deep, 3 points; and an inflammatory cell ring >4 cell layers deep, 4 points.

[0087] Pathological results such as Figure 3AAs shown in the figure, the lung tissue of rats in the CON group had clear alveolar structure, normal septa, minimal inflammatory cell infiltration, and generally normal pathological findings. In the PIC group, alveolar structure disappeared, alveolar septa widened, and numerous inflammatory cells infiltrated the bronchi and peribronchial areas. Compared with the PIC group, the lung tissue pathological findings of the SMZKF-L, SMZKF-M, SMZKF-H, and ASM groups all improved to varying degrees. The improvement effect of SMZKF was dose-dependent, with similar effects in the SMZKF-H and ASM groups.

[0088] Figure 3B and Figure 3C The scores of bronchial inflammation and alveolar inflammation in rats of each group in the embodiment of the present invention are shown respectively. It can be seen that the scores of peribronchial and alveolar inflammation in rats of the PIC group were significantly increased, and the differences were statistically significant ( P <0.05); Compared with the PIC group, the SMZKF-L group, SMZKF-M group, and SMZKF-H group could reduce the peribronchial and alveolar inflammation scores of rats, showing a certain dose-dependent effect ( P <0.05), and the effect of SMZKF-H group was similar to that of compound methoxyphenamine intervention ( P >0.05).

[0089] (4) BALF inflammatory cell count: The rat BALF was collected and centrifuged at 3000 rpm for 10 min (r = 16 cm) at 4°C to collect the cells in the BALF. The cells were then resuspended in phosphate buffered saline (PBS) and mixed thoroughly before cell counting. 10 μL of the cell suspension was then aspirated for smearing. After drying, the slide was fixed in methanol to preserve the cell morphology. After fixation, the slide was immersed in diluted Giemsa stain and stained. The excess dye was removed and the slide was naturally air-dried. The cells were counted under a microscope according to morphological criteria. The percentage of each type of cell was calculated and multiplied by the total number of cells to obtain the inflammatory cell count of each type.

[0090] The results are as follows Figures 4A-4E As shown in the figure, compared with the CON group, the WBC, LYM, NEU, MON and EOS cell counts in the BALF of rats in the PIC group were significantly increased, and the differences were statistically significant ( P <0.05); Compared with the PIC group, the SMZKF-L group, SMZKF-M group, and SMZKF-H group could reduce the WBC, LYM, NEU, and EOS cell counts in BALF, showing a certain dose-dependent manner ( P <0.05), and the effect of SMZKF-H group was similar to that of compound methoxyphenamine intervention ( P >0.05).

[0091] (5) Evaluation of the content of inflammatory factors and cough-inducing factors in rat BALF: The rat BALF to be tested was centrifuged at 4000 r / min for 10 min at 4°C to obtain the supernatant. The sample was added according to the operating steps in the kit instructions, and the content of inflammatory factors IL-1β, IL-6, TNF-α and inflammatory mediators COX-2, PGE2, and TXA2 in the BALF was measured.

[0092] The results are as follows Figures 5A-5F As shown in the figure, compared with the CON group, the BALF levels of IL-1β, TNF-α, IL-6, COX-2, PGE2, and TXA2 in the PIC group were significantly increased, and the differences were statistically significant (P < 0.05); compared with the PIC group, the SMZKF-L group, SMZKF-M group, and SMZKF-H group could reduce the levels of IL-1β, TNF-α, IL-6, COX-2, PGE2, and TXA2 in the BALF ( P <0.05), and showed a certain dose-dependence. The effect of SMZKF-H group was similar to that of compound methoxyphenamine intervention ( P >0.05).

[0093] (6) Detection of oxidative stress indicators in rat lung tissue: Small pieces of tissue from each group of rats were ground into single cell suspensions, and 500 μL of 10 μmol / L DCFH-DA was added. The suspensions were incubated at 37°C for 20 min to allow the probe and cells to fully interact. PBS was used to wash away the DCFH-DA that had not entered the cells, and the ROS expression intensity was detected by flow cytometry. Next, an appropriate amount of preserved lung tissue was taken, and after preparing tissue homogenate, the sample preparation was carried out strictly according to the requirements of the kit, and the MDA and MPO levels of the lung tissue of each group of rats were measured.

[0094] The results are as follows Figures 6A-6C As shown, Figures 6A-6C The ROS, MDA and MPO levels of rats in each group are shown respectively. It can be seen that compared with the PIC group, the SMZKF-L group, SMZKF-M group and SMZKF-H group can reduce the expression of ROS, MDA and MPO in lung tissue (P < 0.05), showing a certain dose-dependency. The effect of the SMZKF-H group is similar to that of the compound methoxyphenamine intervention (P > 0.05).

[0095] (7) Western blot detection of cough sensitivity-related protein expression in rat lung tissue:

[0096] Total protein was extracted from the lung tissues of rats in each group, and the protein concentration was determined by BCA method. After protein denaturation, the cells were subjected to electrophoresis, transfer, blocking, and antibody incubation [antibody dilution ratios were SP (1:1000), TRPV1 (1:1000), NK-1R (1:1000), CGRP (1:1000), P-NF-κB (1:2000), NLRP3 (1:1000), ASC (1:1000), Cleaved-Caspase-1 (1:500), GSDMD-N (1:500), cleaved-IL-1β (1:500), and GAPDH (1:2000)], and the membranes were washed. The expressions of airway sensitivity-related proteins TRPV1, SP, CGRP, and NK1, and cell pyroptosis-related proteins P-NF-κB, NLRP3, ASC, Cleaved-Caspase-1, GSDMD-N, and cleaved-IL-1β in lung tissues were detected by ECL luminescence. Image immunohistochemistry was used to analyze the expression of TRPV1, SP, CGRP, and NK1 in lung tissues, and P-NF-κB, NLRP3, ASC, Cleaved-Caspase-1, GSDMD-N, and cleaved-IL-1β in lung tissues. Semi-quantitative analysis was performed using J software.

[0097] The expression results of airway sensitivity-related proteins in rat lung tissue are as follows Figures 7A-7E As shown in Figure 2, compared with the CON group, the expression of TRPV1, SP, CGRP, and NK1R proteins in the lung tissues of rats in the PIC group was significantly increased, and the differences were statistically significant ( P <0.05); compared with the PIC group, the SMZKF-L group (i.e. Figure 7A Middle L), SMZKF-M group (i.e. Figure 7A Middle M), SMZKF-H group (i.e. Figure 7A The expression of TRPV1, SP, CGRP and NK1R proteins in lung tissues was significantly decreased, showing a dose-dependent manner ( P <0.05), and the effect of SMZKF-H group was similar to that of compound methoxyphenamine intervention ( P >0.05).

[0098] The expression results of pyroptosis-related proteins in rat lung tissue are as follows Figures 8A-8G As shown in the figure, compared with the CON group, the expression of P-NF-κB, NLRP3, ACS, Cleaved-Caspase-1, Cleaved-IL-1β and GSDMD-N proteins in the lung tissue of rats in the PIC group was significantly increased, and the difference was statistically significant ( P <0.05); compared with the PIC group, the SMZKF-L group (i.e. Figure 8A Middle L), SMZKF-M group (i.e. Figure 8A Middle M), SMZKF-H (ie Figure 8AMiddle H) The expression of P-NF-κB, NLRP3, ACS, Cleaved-Caspase-1, Cleaved-IL-1β and GSDMD-N proteins in lung tissues were significantly decreased in a dose-dependent manner ( P <0.05), and the effect of SMZKF-H group was similar to that of compound methoxyphenamine intervention ( P >0.05).

[0099] It can be seen from the above examples that SMZKF can improve airway neurogenic inflammation, reduce cough sensitivity and tracheal inflammation in PIC rats by inhibiting TRPV1-mediated SP / NK1R and CGRP release and cell pyroptosis, which well confirms the scientific nature of SMZKF in preventing and treating PIC and preliminarily reveals the potential molecular mechanism of SMZKF's action.

[0100] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Chinese medicine composition for treating post-infectious cough, characterized in that: It is composed of the following ingredients: 10g of mulberry leaves, 8g of roasted ephedra, 10g of golden fecundation, 10g of honey baiqian, 10g of perilla seeds, 10g of loquat leaves, 10g of burdock seeds, 10g of thunbergia bulb, 10g of Scrophularia ningpoensis, 10g of bombyx batryticatus, 10g of platycodon and 6g of roasted licorice.

2. A method for preparing a Chinese medicine composition, characterized in that: The following steps are involved: Soaking the various raw materials in the traditional Chinese medicine composition of claim 1 in water for 80-100 minutes, boiling and then decocting for 60-120 minutes, filtering to obtain a decoction and a residue; The residue is soaked in water, boiled and then decocted for 30-50 minutes, filtered to obtain a decoction, and the two decoctions are combined and concentrated to obtain a traditional Chinese medicine composition.

3. The method for preparing the Chinese medicine composition according to claim 2, wherein The mass volume ratio of the traditional Chinese medicine composition to distilled water is 1:5-20 g / L; The mass volume ratio of the residue to distilled water is 0.5-1.5:8 g / L.

4. A Chinese medicine preparation, characterized in that Contains the Chinese medicine composition according to claim 1.

5. The Chinese medicine preparation according to claim 4, characterized in that The preparation types of the traditional Chinese medicine preparation include tablets, capsules, granules, pills, decoctions or powders.

6. Use of the Chinese medicine composition according to claim 1 in preparing medicine for treating post-infectious cough.

7. The use according to claim 6, characterized in that The traditional Chinese medicine composition improves post-infectious cough by simultaneously acting on the SP / NK1R and CGRP release mediated by TRPV1 and the cell pyroptosis pathway.

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