Application of traditional Chinese medicine composition in preparation of medicine for treating or preventing severe pneumonia

Abstract

The invention relates to the technical field of traditional Chinese medicines, and particularly discloses application of a traditional Chinese medicine composition in preparation of a medicine for treating or preventing severe pneumonia. Researches prove that the traditional Chinese medicine composition can relieve lung injury and inflammation of a severe pneumonia mouse model, improve lung cell factor level abnormity and reverse immune disorder caused by activation of macrophages and depletion of lymphocytes of the severe pneumonia mouse. The traditional Chinese medicine composition can protect lung capillaries and prevent severe pneumonia. Clinical tests prove that all the medicinal materials of the traditional Chinese medicine composition act together, and the traditional Chinese medicine composition shows a remarkable clinical curative effect on severe pneumonia patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly relates to application of a traditional Chinese medicine composition in preparation of a medicine for treating or preventing severe pneumonia. BACKGROUND

[0002] Severe pneumonia is a respiratory critical illness caused by progressive development of community-acquired pneumonia or hospital-acquired pneumonia, eventually leading to multiple organ failure mainly in lung injury, and the mortality of inpatient cases can reach 30% to 50%, which constitutes a major risk to the elderly, children and individuals with low immunity.

[0003] The clinical symptoms of severe pneumonia include high fever, cough, shortness of breath, and in severe cases, blurred consciousness, hypotension and other physical signs. The pathophysiological characteristics are the complex interaction between pathogen invasion and host immune response, leading to severe lung and systemic injury. The central link of this process is the replication of pathogens in the lower respiratory tract, causing direct lung injury and triggering a strong and complex immune response. The body is in a complex state of immune imbalance, including immune suppression and immune overreaction. In elderly patients, there may also be problems such as immune aging and immune paralysis. While general pneumonia is mainly an immune overreaction inflammatory response under the stimulation of pathogens, the immune state is relatively simple. The diagnostic criteria for severe pneumonia and general pneumonia are different, and the treatment regimens are also different. The medication for severe pneumonia is not simply anti-inflammatory treatment. For example, glucocorticoids have strong anti-inflammatory and anti-immune pharmacological effects. However, a 2016 study on patients with severe H7N9 pneumonia showed that patients using high-dose (> 150 mg / d) methylprednisolone or equivalent hormones had significantly higher 30-day and 60-day mortality rates, and the viral carrying time in the body was significantly prolonged, indicating that the use of glucocorticoids actually increased the mortality rate. This suggests that severe pneumonia is significantly different from ordinary inflammation, and the choice of medication should be based on the specific immune state of the body.

[0004] Traditional Chinese medicine has been increasingly widely used in immune regulation. Some traditional Chinese medicines can enhance the body's cellular and humoral immune functions, promote the physiological functions of lymphocytes, mononuclear macrophages and hematopoietic stem cells; some traditional Chinese medicines have immune suppression function, can reduce the release of inflammatory factors, inhibit or eliminate the production of antibodies, inhibit the proliferation of T cells, etc., and prevent and treat the damage caused by excessive immune response to the body. However, for the complex immune environment of severe pneumonia, it is not simply to promote or inhibit immunity, so not all traditional Chinese medicines with corresponding immune regulation effects are effective. Therefore, it is of great significance to develop traditional Chinese medicines that can be used in complex immune environments to treat severe pneumonia. SUMMARY

[0005] Based on the above technical status, the purpose of the present application is to provide application of a traditional Chinese medicine composition in preparation of a medicine for treating severe pneumonia.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] A traditional Chinese medicine composition is used in preparing a medicine for treating severe pneumonia. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 52-86 parts of ephedra; 194-324 parts of gypsum; 194-324 parts of forsythia; 78-130 parts of scutellaria; 194-324 parts of mulberry bark; 78-130 parts of bitter almond; 78-130 parts of peucedanum; 78-130 parts of pinellia; 78-130 parts of tangerine peel; 78-130 parts of fritillaria; 78-130 parts of burdock fruit; 78-130 parts of honeysuckle; 39-65 parts of rhubarb; 46-76 parts of platycodon; and 39-65 parts of liquorice.

[0008] Furthermore, the Chinese medicine composition is prepared from the following raw materials in parts by weight: 52 parts of ephedra; 324 parts of gypsum; 194 parts of forsythia; 78 parts of scutellaria; 194 parts of mulberry bark; 130 parts of bitter almond; 78 parts of peucedanum; 130 parts of pinellia; 78 parts of tangerine peel; 78 parts of fritillaria; 130 parts of burdock fruit; 130 parts of honeysuckle; 39 parts of rhubarb; 76 parts of platycodon; and 65 parts of licorice.

[0009] Furthermore, the Chinese medicine composition is prepared from the following raw materials in parts by weight: 86 parts of ephedra; 194 parts of gypsum; 324 parts of forsythia; 130 parts of scutellaria; 324 parts of mulberry bark; 78 parts of bitter almond; 130 parts of peucedanum; 78 parts of pinellia; 130 parts of tangerine peel; 130 parts of fritillaria; 78 parts of burdock fruit; 78 parts of honeysuckle; 65 parts of rhubarb; 46 parts of platycodon; and 39 parts of licorice.

[0010] Furthermore, the Chinese medicine composition is prepared from the following raw materials in parts by weight: 69 parts of ephedra; 259 parts of gypsum; 259 parts of forsythia; 104 parts of scutellaria; 259 parts of mulberry bark; 104 parts of bitter almond; 104 parts of peucedanum; 104 parts of pinellia; 104 parts of tangerine peel; 104 parts of fritillaria; 104 parts of burdock fruit; 104 parts of honeysuckle; 52 parts of rhubarb; 61 parts of platycodon; and 52 parts of licorice.

[0011] Furthermore, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 55 parts of ephedra; 254 parts of gypsum; 318 parts of forsythia; 107 parts of scutellaria; 203 parts of mulberry bark; 107 parts of bitter almond; 82 parts of peucedanum; 105 parts of pinellia; 84 parts of tangerine peel; 125 parts of fritillaria; 122 parts of burdock fruit; 113 parts of honeysuckle; 42 parts of rhubarb; 60 parts of platycodon; and 50 parts of licorice.

[0012] Furthermore, the bitter almonds are fried bitter almonds, the Fritillaria is Fritillaria thunbergii, the honeysuckle is Lonicera japonica, and the pinellia is Pinellia ternata.

[0013] The animal experiment proves that the traditional Chinese medicine composition can reduce the lung injury and inflammation of the severe pneumonia mouse model, improve the abnormal cytokine level in the lung, and reverse the immune disorder caused by the macrophage activation and lymphocyte depletion of the severe pneumonia mouse.

[0014] The active ingredient of the traditional Chinese medicine composition is prepared by the following steps:

[0015] A. The beimu is weighed according to the prescription proportion, crushed into fine powder, and reserved;

[0016] B. The mahuang, lianqiao, kuaxingren, banxia, niubangzi, and dahuang are weighed according to the prescription proportion, and 40-70% ethanol is used for reflux extraction for 2 times, 1-4 hours each time, 8-10 times for the first time, and 6-9 times for the second time. The extraction liquid is combined and filtered. The filtrate is concentrated under reduced pressure to recover ethanol. The concentrated filtrate is heated to 60 DEG C, and the relative density is 1.14-1.16. The clear paste is reserved.

[0017] C. The shigao, sangbaipi, qianhu, chenpi, jinyinhua, jiegeng, and gancao are weighed according to the prescription proportion, and water is used for decoction for 2 times, 1-4 hours each time, 9-11 times for the first time, and 7-9 times for the second time. The decoction liquid is combined and filtered. The concentrated filtrate is heated to 60 DEG C, and the relative density is 1.14-1.16. The clear paste is combined with the clear paste obtained in step B, and reserved.

[0018] The fine powder obtained in step A and the combined clear paste obtained in step C jointly constitute the active ingredient of the drug composition.

[0019] The dosage form of the drug can be a capsule, a tablet, a powder, a granule, an oral liquid, a soft capsule, a pill, a tincture, a syrup, a suppository, a gel, a spray, or an injection.

[0020] To enable the above-mentioned dosage forms, pharmaceutically acceptable adjuvants, such as fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, etc. are added when preparing these dosage forms. Fillers include starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methyl cellulose, etc.; binders include starch paste, polyvinylpyrrolidone, hydroxypropyl methyl cellulose, etc.; sweeteners include sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include sweeteners and various flavors; preservatives include nipagin, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil, etc.; bases include PEG6000, PEG4000, insect wax, etc.

[0021] The tablet is prepared by the following steps:

[0022] A. Fritillaria is weighed according to the prescription ratio, crushed into fine powder, and reserved;

[0023] B. Ephedra, forsythia, bitter apricot kernel, pinellia, burdock fruit, and rhubarb are weighed according to the prescription ratio, extracted with 40-70% ethanol for 2 times, 1-4 hours each time, 8-10 times the amount is added in the first time, and 6-9 times the amount is added in the second time. The extraction liquid is combined and filtered. The filtrate is concentrated under reduced pressure to a clear paste with a relative density of 1.14-1.16 at 60°C. The clear paste is reserved.

[0024] C. Gypsum, mulberry bark, peony root, dried tangerine or orange peel, honeysuckle, platycodon root, and licorice are weighed according to the prescription ratio, decocted with water for 2 times, 1-4 hours each time, 9-11 times the amount is added in the first time, and 7-9 times the amount is added in the second time. The decoction is combined and filtered. The clear paste is concentrated to a clear paste with a relative density of 1.14-1.16 at 60°C. The clear paste is combined with the clear paste obtained in step B, and reserved.

[0025] D. The combined clear paste obtained in step C is spray dried, and the spray powder is collected and reserved.

[0026] E. The spray powder obtained in step D and the fine powder obtained in step A are mixed with ethanol as a binder to prepare a soft material, sieved and granulated. The granules are compressed to obtain tablets according to the conventional method of pharmacy.

[0027] The drug for treating severe pneumonia is preferably a drug that reduces the levels of pro-inflammatory factors and anti-inflammatory factors in the body of a patient with severe pneumonia.

[0028] Further, the pro-inflammatory factors include IL6, IL-1β and / or TNF-α, and the anti-inflammatory factors include IL-4, IL10 and / or FOXP3.

[0029] The drug for treating severe pneumonia is preferably a drug for simultaneously adjusting immune hyperactivity and immune suppression in a patient with severe pneumonia.

[0030] Further, the drug is a drug for improving macrophage activation and lymphocyte depletion in a patient with severe pneumonia. The drug reduces the proportion of macrophages, increases the proportion of B cells, CD4 + T cells and CD8 + T cells in the patient with severe pneumonia.

[0031] Further, the drug is used for preparing a drug for protecting lung microvessels.

[0032] Further, the drug can improve lung microvascular endothelial cell viability, reduce lung microvascular endothelial cell death, and protect lung microvascular endothelial cell barrier function.

[0033] Further, the drug can improve the transendothelial electrical resistance value of lung microvascular endothelial cells, and improve the expression of VE-cadherin and Occludin proteins of lung microvascular endothelial cells. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Volcano plot analysis of differentially expressed proteins in lung tissues of each group determined by Olink method;

[0035] Figure 2 GO function and KEGG pathway enrichment analysis of mouse lung tissues;

[0036] Figure 3 Heat map analysis of differentially expressed proteins in lung tissues of each group determined by Olink method;

[0037] Figure 4 CyTOF technology for observing the classification of main immune cells in mouse lung tissues;

[0038] Figure 5 CyTOF technology for observing the number of various immune cells in lung tissues of each group;

[0039] Figure 6 CyTOF technology for observing the subtypes of different immune cells in lung tissues of each group;

[0040] Figure 7 CyTOF technology for observing CD4 + cell subtypes in lung tissues of each group;

[0041] Figure 8To observe the CD8 + cell typing in each group of lung tissues by CyTOF technology;

[0042] Figure 9 To observe the granulocyte typing in each group of lung tissues by CyTOF technology;

[0043] Figure 10 To observe the macrophage typing in each group of lung tissues by CyTOF technology. DETAILED DESCRIPTION

[0044] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application.

[0045] Example 1

[0046] Prescription: Ephedra 52g; Gypsum 324g; Forsythia 194g; Scutellaria 78g; White Mulberry Bark 194g; Fried Bitter Almond 130g; Peucedanum 78g; Pinellia 130g; Tangerine Peel 78g; Zhejiang Fritillaria 78g; Burdock Achene 130g; Honeysuckle 130g; Rhubarb 39g; Platycodon 76g; Licorice 65g.

[0047] Preparation method: A, Zhejiang Fritillaria was weighed according to the prescription proportion, crushed into fine powder, and reserved for use; B, Ephedra, Forsythia, Fried Bitter Almond, Pinellia, Burdock Achene, and Rhubarb were weighed according to the prescription proportion, and extracted with 50% ethanol by reflux extraction for 2 times, 3 hours each time, 10 times the amount was added for the first time, and 6 times the amount was added for the second time, the extraction liquid was combined and filtered, the filtrate was recovered by reducing pressure, and the ethanol was concentrated to a clear extract with a relative density of 1.15 at 60°C, and reserved for use; C, Gypsum, White Mulberry Bark, Peucedanum, Tangerine Peel, Honeysuckle, Platycodon, and Licorice were weighed according to the prescription proportion, and boiled in water for 2 times, 2 hours each time, 10 times the amount was added for the first time, and 7 times the amount was added for the second time, the decoction was combined and filtered, the filtrate was recovered by reducing pressure, and the ethanol was concentrated to a clear extract with a relative density of 1.15 at 60°C, and combined with the clear extract obtained in step B, and reserved for use; D, the combined clear extract obtained in step C was spray dried, and the spray powder was collected and reserved for use; E, the weight ratio of the raw materials required for tabletting was as follows: spray powder obtained in step D 282.6; fine powder obtained in step A 101; carboxymethyl starch sodium 12.5; microcrystalline cellulose 9.0; magnesium stearate 2.25, starch was added, and the tablet was prepared by the conventional preparation method, and thus the tablet was obtained.

[0048] Example 2

[0049] Ingredients: Ephedra 86g; Gypsum 194g; Forsythia 324g; Scutellaria 130g; White Mulberry Bark 324g; Fried Bitter Almond 78g; Peucedanum 130g; Pinellia 78g; Tangerine Peel 130g; Fritillaria 130g; Burdock Achene 78g; Honeysuckle 78g; Rhubarb 65g; Platycodon 46g; Licorice 39g.

[0050] Preparation: A, according to the proportion of the prescription, Fritillaria was weighed and ground into fine powder for later use; B, according to the proportion of the prescription, Ephedra, Forsythia, Fried Bitter Almond, Pinellia, Burdock Achene and Rhubarb were weighed and extracted with 40% ethanol for 2 times, 4 hours each time, 8 times the amount for the first time and 9 times the amount for the second time. The extract was combined and filtered. The filtrate was concentrated to a clear extract with a relative density of 1.14 at 60°C, and was ready for use; C, according to the proportion of the prescription, Gypsum, White Mulberry Bark, Peucedanum, Tangerine Peel, Honeysuckle, Platycodon and Licorice were weighed and decocted with water for 2 times, 4 hours each time, 9 times the amount for the first time and 7 times the amount for the second time. The decoction was combined, filtered, and concentrated to a clear extract with a relative density of 1.16 at 60°C. The extract was combined with the extract from step B, and was ready for use; D, the combined extract from step C was spray-dried, and the spray-dried powder was collected for later use; E, the weight ratio of the required materials for tabletting was as follows: spray-dried powder from step D 200; fine powder from step A 145; carboxymethyl starch sodium 10; microcrystalline cellulose 12; and magnesium stearate 1.5. The tablet was prepared according to the conventional preparation method, and was ready.

[0051] Example 3

[0052] Ingredients: Ephedra 86g; Gypsum 194g; Forsythia 324g; Scutellaria 130g; White Mulberry Bark 324g; Fried Bitter Almond 78g; Peucedanum 130g; Pinellia 78g; Tangerine Peel 130g; Fritillaria 130g; Burdock Achene 78g; Honeysuckle 78g; Rhubarb 65g; Platycodon 46g; Licorice 39g.

[0053] Preparation method: A, according to the proportion of the prescription Zhebeimu, crushed into fine powder, ready for use; B, according to the proportion of the prescription of Ephedra, forsythia, Fried bitter almonds, pinellia, arctium lappa, rhubarb plus 70% ethanol reflux extraction 2 times, 1 hour each time, the first time plus 10 times, the second time plus 6 times, the extraction was combined, filtered, the filtrate was recovered by reducing pressure, concentrated to 60 ℃ heat measuring relative density of 1.16 clear paste, ready for use; C, according to the proportion of the prescription of gypsum, mulberry bark, radix peucedani, dried tangerine or orange peel, honeysuckle, platycodon grandiflorum, licorice, plus water decocting two times, 1 hour each time, the first time plus 11 times, the second time plus 7 times, the decoction was combined, filtered, the filtrate was recovered by reducing pressure, concentrated to 60 ℃ heat measuring relative density of 1.14 clear paste, with the clear paste obtained in step B combined, ready for use; D, the combined clear paste obtained in step C, spray drying, collecting spray powder ready for use; E, the weight parts of the raw materials required for tabletting are as follows: 355 of the spray powder obtained in step D; 54 of the fine powder obtained in step A; 15.5 of carboxymethyl starch sodium; 7 of microcrystalline cellulose; 3.5 of magnesium stearate, add starch, according to the conventional preparation method to make tablets, ready.

[0054] Example 4

[0055] The raw material formula is: Ephedra 55g; Gypsum 254g; forsythia 318g; radix scrophulariae 107g; mulberry bark 203g; Fried bitter almonds 107g; radix peucedani 82g; pinellia 105g; dried tangerine or orange peel 84g; Zhebeimu 125g; arctium lappa 122g; honeysuckle 113g; rhubarb 42g; platycodon grandiflorum 60g; licorice 50g.

[0056] Preparation method: A, according to the proportion of the prescription Zhebeimu, crushed into fine powder, ready for use; B, according to the proportion of the prescription of Ephedra, forsythia, Fried bitter almonds, pinellia, arctium lappa, rhubarb plus 70% ethanol reflux extraction 2 times, 1 hour each time, the first time plus 10 times, the second time plus 6 times, the extraction was combined, filtered, the filtrate was recovered by reducing pressure, concentrated to 60 ℃ heat measuring relative density of 1.16 clear paste, ready for use; C, according to the proportion of the prescription of gypsum, mulberry bark, radix peucedani, dried tangerine or orange peel, honeysuckle, platycodon grandiflorum, licorice, plus water decocting two times, 1 hour each time, the first time plus 11 times, the second time plus 7 times, the decoction was combined, filtered, the filtrate was recovered by reducing pressure, concentrated to 60 ℃ heat measuring relative density of 1.14 clear paste, with the clear paste obtained in step B combined, ready for use; D, the combined clear paste obtained in step C, spray drying, collecting spray powder ready for use; E, the weight parts of the raw materials required for tabletting are as follows: 355 of the spray powder obtained in step D; 54 of the fine powder obtained in step A; 15.5 of carboxymethyl starch sodium; 7 of microcrystalline cellulose; 3.5 of magnesium stearate, add starch, according to the conventional preparation method to make tablets, ready.

[0057] Example 5

[0058] Ephedra 62g; Gypsum 220g; Forsythia 256g; Scutellaria 90g; White Mulberry Bark 300g; Bitter Apricot Kernel 90g; Peucedanum 90g; Pinellia 90g; Tangerine Peel 100g; Fritillaria 100g; Burdock Achene 100g; Honeysuckle 100g; Rhubarb 50g; Platycodon 66g; Licorice 50g.

[0059] The above medicinal materials are made into capsules by a conventional method.

[0060] Example 6:

[0061] Ephedra 68g; Gypsum 215g; Forsythia 215g; Scutellaria 100g; White Mulberry Bark 220g; Bitter Apricot Kernel 90g; Peucedanum 90g; Pinellia 90g; Tangerine Peel 90g; Fritillaria 90g; Burdock Achene 90g; Honeysuckle 90g; Rhubarb 50g; Platycodon 50g; Licorice 50g.

[0062] The above medicinal materials are made into granules by a conventional method.

[0063] Example 7:

[0064] Ephedra 50g; Gypsum 200g; Forsythia 300g; Scutellaria 100g; White Mulberry Bark 250g; Bitter Apricot Kernel 100g; Peucedanum 100g; Pinellia 100g; Tangerine Peel 100g; Fritillaria 100g; Burdock Achene 100g; Honeysuckle 100g; Rhubarb 50g; Platycodon 50g; Licorice 50g.

[0065] The above medicinal materials are made into injections by a conventional method.

[0066] Example 8:

[0067] Ephedra 60g; Gypsum 200g; Forsythia 200g; Scutellaria 95g; White Mulberry Bark 230g; Bitter Apricot Kernel 95g; Peucedanum 95g; Pinellia 95g; Tangerine Peel 95g; Fritillaria 95g; Burdock Achene 95g; Honeysuckle 95g; Rhubarb 50g; Platycodon 50g; Licorice 50g.

[0068] The above medicinal materials are made into pills by a conventional method.

[0069] To clarify the effect of the medicine of the present application on severe pneumonia, the following test study was conducted using the tablets (LHQK tablets) prepared according to the method of Example 1.

[0070] Test Example 1 Clinical Efficacy of LHQK on Severe Pneumonia Patients

[0071] I. Materials and Methods

[0072] 1. Clinical Data

[0073] Eighty patients with severe pneumonia admitted to the Second Hospital of Hebei Medical University from February 2023 to December 2023 were selected as the research objects. Before the formal start of the clinical research, it was reported to the Ethics Committee of the Second Hospital of Hebei Medical University. All procedures in this study were conducted in accordance with the ethical guidelines of the Helsinki Declaration. The ethical review number was 2023-C008-F1. And completed registration in the China Clinical Trial Registry, registration number: ChiCTR2300067998.

[0074] 2. Selection of subjects

[0075] 2.1 Diagnostic criteria

[0076] The diagnostic criteria for severe pneumonia refer to the "Guidelines for the Diagnosis and Treatment of Adult Community-acquired Pneumonia in China (2016 Edition)". Those who meet the following 1 major criterion or ≥3 secondary criteria can be diagnosed as severe pneumonia.

[0077] 2.1.1 Major criteria:

[0078] (1) Need for endotracheal intubation for mechanical ventilation therapy;

[0079] (2) Septic shock still requires vasopressor therapy after active fluid resuscitation.

[0080] 2.1.2 Secondary criteria:

[0081] (1) Respiratory rate ≥30 times / min;

[0082] (2) OI≤250mmHg (1mmHg=0.133kPa);

[0083] (3) Multiple lobe infiltration;

[0084] (4) Consciousness disorder and / or disorientation;

[0085] (5) Blood urea nitrogen ≥7.14mmol / L;

[0086] (6) Systolic blood pressure <90mmHg requires active fluid resuscitation.

[0087] 2.2 Inclusion criteria

[0088] (1) Meet the diagnostic criteria for severe pneumonia;

[0089] (2) 18 years old ≤ age ≤ 80 years old;

[0090] (3) Diagnosed within 48 hours of severe pneumonia;

[0091] 2.3 Exclusion criteria

[0092] (1) Patients with severe heart failure, liver and kidney failure;

[0093] (2) The patient's expected survival time is less than 14 days due to underlying diseases, such as poorly controlled malignant tumors or cardiac arrest within the past 30 days;

[0094] (3) patients with severe mental illness;

[0095] (4) Those who are pregnant or trying to become pregnant, or those who are unable to take effective contraceptive measures;

[0096] (5) Allergy to the active ingredients of the test drug or its excipients;

[0097] (6) Participated in a clinical intervention trial in the past 3 months;

[0098] (7) Subjects who are deemed unsuitable to participate in this trial by the attending physician.

[0099] 3. Study Design

[0100] A randomized, open, parallel controlled clinical research method was used.

[0101] (1) Randomization: The medical statistician was responsible for generating a random number table using SAS software and randomly dividing the patients into a low-dose LHQK tablet group, a high-dose LHQK tablet group, and a basic treatment group in a ratio of 1:1:2.

[0102] (2) Control: High-dose group, low-dose group and basic treatment group were used as controls.

[0103] (3) Sample size and grouping: A total of 80 cases, 20 cases in the low-dose group, 20 cases in the high-dose group, and 40 cases in the basic treatment group.

[0104] 4. Treatment options

[0105] 4.1 Name, strength and batch number of study drug

[0106] LHQK tablets, national medicine approval number Z20200004, specification 0.46g / tablet, produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd.

[0107] 4.2 Distribution, Recovery, and Storage of Research Drugs

[0108] (1) Distribution and recovery of research drugs: Drugs are distributed to subjects by the research center's drug management personnel. Records should be kept for each drug distribution process.

[0109] (2) Storage of experimental drugs: Establish a management system for experimental drugs during the trial period, set up a special counter to keep the experimental drugs, and store them in a ventilated and dry place, and be managed uniformly by the experimental drug manager.

[0110] 4.3 Dosage

[0111] 4.3.1 Dosing Regimen

[0112] Basic treatment group: basic treatment

[0113] Low-dose group: basic treatment + LHQK tablets, oral, 4 tablets / time, 3 times / day;

[0114] High-dose group: basic treatment + LHQK tablets, oral, 8 tablets / time, 3 times / day;

[0115] 4.3.2 Basic treatment (2016 Chinese Expert Consensus on Clinical Practice of Severe Pneumonia)

[0116] (1) Principles for the use of antibacterial drugs: Patients with severe pneumonia should be given appropriate empirical initial antibacterial drug treatment immediately, and pathogenic detection samples should be taken before antibacterial drug treatment. Based on clinical and epidemiological basis, the antibacterial drug regimen should cover as many possible pathogens as possible. When the pathogen of severe pneumonia is not clear, broad-spectrum antibacterial drug treatment is recommended.

[0117] Anti-infective therapy can generally be stopped 3-5 days after defervescence and significant improvement of major respiratory symptoms, but the course of treatment varies depending on the different pathogens and the severity of the disease. Complete absorption of lung shadows cannot be used as an indicator for stopping the use of antibacterial drugs. For common bacterial infections such as Streptococcus pneumoniae, the drug can be used for 72 hours after defervescence. For infections caused by pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella or anaerobes, which can easily cause lung tissue necrosis, the recommended course of antibacterial drug treatment is ≥2 weeks. The course of treatment for patients with atypical pathogens who respond more slowly can be extended to 10-14 days. The recommended course of treatment for Legionella infection is 10-21 days. Although some studies have shown that a course of 7 days or more does not seem to increase clinical efficacy and improve clinical outcomes, these studies are mostly outpatient patients or patients who have a rapid response to initial treatment. For severe pneumonia, especially patients who have poor initial response or even failure, the treatment course will be significantly prolonged.

[0118] (2) Glucocorticoids: It is recommended that community-acquired pneumonia patients with septic shock can follow the principles of septic shock treatment and use small doses of glucocorticoids for a short period of time. For patients with severe pneumonia who do not have septic shock, the use of glucocorticoids is not routinely recommended.

[0119] (3) Gamma globulin: It is widely used in clinical practice and has certain clinical effects, and its effect on immunodeficient patients and viral infections should be affirmed. There is still controversy about bacterial infections, and the clinical efficacy of patients with severe pneumonia caused by bacterial infections needs further evidence-based evidence.

[0120] (4) Symptomatic support treatment: including albumin, nutritional support, oxygen therapy and assisted breathing, and promoting respiratory tract secretions drainage, etc.

[0121] 5. Observation index

[0122] 5.1 Evaluation of clinical effectiveness of LHQK tablets on patients with severe pneumonia

[0123] 5.1.1 Primary clinical efficacy index:

[0124] Clinical comprehensive efficacy evaluation: change value and change rate of Acute Physiology and Chronic Health Evaluation-II (APACHE-II) score after 7 days of treatment

[0125] 5.1.2 Secondary clinical efficacy index:

[0126] (1) Total length of hospital stay;

[0127] (2) Change degree of chest X-ray after 7 days of treatment.

[0128] (3) Airway epithelial mucosa: pathological analysis, proportion of ciliated cells, MUC5AC protein content;

[0129] (4) Sputum: viscosity detection;

[0130] (5) Alveolar lavage fluid: proportion of inflammatory cells

[0131] 5.2 Evaluation of clinical safety of LHQK tablets on patients with severe pneumonia

[0132] (1) Adverse events;

[0133] (2) General physical examination items, such as body temperature, heart rate, respiration, etc.

[0134] (3) Blood routine, C-reactive protein, urine routine, liver function, kidney function, electrocardiogram.

[0135] 6. Research process

[0136] 6.1 Observation items and observation time points

[0137] 6.1.1 Baseline (V1, -1 to 0 days)

[0138] (1) Blood routine (white blood cells, red blood cells, neutrophils, lymphocytes, platelets, hemoglobin), C-reactive protein, urine routine, liver function, kidney function.

[0139] (2) Arterial blood gas analysis, electrolytes

[0140] (3) Chest X-ray, electrocardiogram

[0141] (4) Specimen collection: Bronchoalveolar lavage fluid, sputum, airway mucosa

[0142] (5) Physical examination / vital signs / clinical symptoms

[0143] (6) Review of inclusion criteria, exclusion criteria

[0144] (7) Medical history, demographic data, record of concomitant medication

[0145] (8) Signing of informed consent, randomization, dispensing of study drug

[0146] 6.1.2 Visit 2 (V2, 7 days after medication or treatment end ± 1 day)

[0147] (1) Blood routine (white blood cell, red blood cell, neutrophil, lymphocyte, platelet, hemoglobin), C-reactive protein, urine routine, liver function, kidney function.

[0148] (2) Arterial blood gas analysis, electrolyte

[0149] (3) Chest X-ray, electrocardiogram

[0150] (4) Specimen collection: Bronchoalveolar lavage fluid, sputum, airway mucosa

[0151] (5) Physical examination / vital signs / clinical symptoms

[0152] (6) Record of concomitant medication

[0153] 6.2 Efficacy index

[0154] 6.2.1 Primary efficacy index:

[0155] APACHE-Ⅱ score table score: Calculate the change value and change rate of APACHE-Ⅱ score after 7 days of treatment or after treatment end ± 1 day compared with baseline period

[0156] 6.2.2 Secondary efficacy index:

[0157] (1) Total length of hospitalization

[0158] Calculate the total length of hospitalization of patients, the formula is: total length of hospitalization = discharge date - hospitalization date

[0159] (2) Chest X-ray change degree

[0160] Calculate the change degree of chest X-ray after 7 days of treatment or after treatment end ± 1 day compared with baseline period

[0161] (3) Airway epithelial mucosa: pathological analysis, cilia cell ratio, MUC5AC protein content;

[0162] (4) Sputum: Viscosity test;

[0163] (5) Alveolar lavage fluid: Inflammatory cell ratio

[0164] 7. Materials and methods

[0165] 7.1 Experimental reagents

[0166] 4% paraformaldehyde solution Wuhan Sivier Biological Technology Co., Ltd.

[0167] Hematoxylin / Eosin staining solution Shanghai Biyun Tian Biological Technology Co., Ltd.

[0168] Wright's Giemsa staining solution Zhuhai Baisuo Biological Technology Co., Ltd.

[0169] Neutral gum sealing agent Beijing Solapure Biological Technology Co., Ltd.

[0170] MUC5AC antibody USA Abeam Company

[0171] β-tubulin antibody USA Abeam Company

[0172] Goat anti-rabbit IgG H&L (HRP) USA Abeam Company 594)

[0173] Antibody diluent Wuhan Yakeyin Biological Technology Co., Ltd.

[0174] Universal two-step kit Beijing Zhongsu Jinqiao Biological Technology Co., Ltd.

[0175] DAB solution Beijing Zhongsu Jinqiao Biological Technology Co., Ltd.

[0176] EDTA solution Beijing Solapure Biological Technology Co., Ltd.

[0177] DAPI Shanghai Biyun Tian Biological Technology Co., Ltd.

[0178] 7.2 Experimental instruments

[0179] RM 2235 microtome Germany Leica Company

[0180] Tissue embedding machine Japan Sahura Company

[0181] Tissue section staining machine Japan Sahura Company

[0182] RS-28 type high-speed low-temperature centrifuge Germany Heraeus Company

[0183] High-speed refrigerated centrifuge Wuhan Gene Technology Co., Ltd.

[0184] Large high-speed low-temperature centrifuge USA Beckman Company

[0185] Leica DMi8 inverted fluorescence microscope Leica, Germany

[0186] Leica DM 6000B microscope Leica, Germany

[0187] Smear machine Shanghai Aipudi Experimental Instrument Manufacturing Co., Ltd.

[0188] Digital viscometer Shanghai Sheryan Instrument Co., Ltd.

[0189] 7.3 Sampling content and method

[0190] 7.3.1 Airway mucosa:

[0191] (1) Sampling method: For each case included, use biopsy forceps to take 1 small rice-sized airway mucosa epithelium at the bifurcation of the trachea;

[0192] (2) Storage and transportation: After taking the airway mucosa, put it into a specimen bottle containing 4% paraformaldehyde, and transport at room temperature for 48 hours.

[0193] 7.3.2 Sputum:

[0194] (1) Sampling method: Use a sputum suction tube to aspirate sputum samples near the pulmonary lesion, and the amount of sputum suctioned is 5 mL;

[0195] (2) Storage and transportation: After sample collection, place it in a 4°C incubator with ice bags or ice blocks and transport to the laboratory for sputum viscosity determination.

[0196] 7.3.3 Bronchoalveolar lavage fluid (BALF):

[0197] (1) Sampling method: The specimen for microbiological examination must strictly follow aseptic operation, and the amount of bronchoalveolar lavage fluid taken for each sample is 10 mL (as much as possible to ensure that the bronchoalveolar lavage fluid is injected consistently), placed in a 4°C incubator with ice bags, and sent to the laboratory within the shortest time (not more than 4 hours), the amount of lavage fluid for each sample is kept consistent, thereby ensuring the consistent dilution ratio of inflammatory cells.

[0198] (2) Storage and transportation: After sample collection, place it in a 4°C incubator with ice bags or ice blocks and transport to the laboratory for subsequent experiments.

[0199] 7.4 Experimental method

[0200] 7.4.1 Hematoxylin and Eosin staining (H&E)

[0201] The patient's airway mucosa was put into an embedding box and soaked in paraformaldehyde for 48 hours for fixation, followed by routine alcohol dehydration, xylene transparency, and paraffin embedding. After 4 pm sectioning, HE staining was performed. The specific steps are as follows: 60°C constant temperature oven baking 5h→xylene dewaxing, sequentially immersed in xylene (I) 30min→xylene (II) 30min→100% ethanol 2min→95% ethanol 3min→90% ethanol 3min→60% ethanol 3min→distilled water washing 2min; 5min of hematoxylin restaining, slow tap water rinse; 1% hydrochloric acid-ethanol differentiation 10s, running water rinse to return blue; 5min of eosin staining, water washing 3 times; neutral gum mounting; observed under a microscope and collected images, and finally analyzed the pathological changes.

[0202] 7.4.2 Immunofluorescence

[0203] After the patient's airway mucosa pathological section was routinely dewaxed to water, microwave heat repair was performed with EDTA sodium citrate antigen repair solution. After cooling to room temperature, PBS was used for washing 3 times, each for 5 min; 3% hydrogen peroxide solution was added dropwise and incubated at 37°C in the dark for 10 min. The section was washed with PBS 3x5min and blocked with 5% BSA at room temperature for 30 min; the tissue was incubated with β-tubulin IV (β-tubulin IV) antibody (Abeam, ab179509, 1:500 dilution) at 4°C overnight; the next day, the section was washed with PBS 3x5min, and the tissue was incubated with Alexa Fluor 594 (Abeam, ab150064, 1:500 dilution) at room temperature for 1 hour, and then washed with PBS 3x5min; finally, DAPI was added to stain the cell nucleus and the section was mounted. Images were captured using a microscope. Image software was used to automatically identify the target protein and calculate its pixel area.

[0204] 7.4.3 Immunohistochemistry

[0205] The patient's airway mucosa section was dewaxed and microwave heat repaired with sodium citrate antigen repair solution. After cooling to room temperature, PBS was used for washing 3 times, each for 5 min; then endogenous peroxidase blocker was added and incubated at room temperature for 10 min, and washed with PBS 3x5min; the section was incubated with Mucin 5AC (MUC5AC) (Abeam, ab3649, 1:500 dilution) antibody for 60 min, and washed with PBS 3x5min; then goat anti-mouse / rabbit IgG polymer was added and incubated for 20 min, and washed with PBS 3x5min, and then DAB substrate was used for 3 min of reaction; after DAB color development, the staining was immediately terminated, the cell nucleus was restained with hematoxylin, and the section was mounted. Under a microscope, the target antigen of the target cell was brownish yellow, which was positive expression. ImageJ software was used to automatically identify the target protein and calculate its pixel area.

[0206] 7.4.4 Wright-Giemsa staining

[0207] The recovered BALF was first filtered with a single layer of gauze to remove mucus, centrifuged at 400g for 10 min, and the supernatant and cells were collected separately, of which the supernatant was aliquoted and stored at -80°C for later use; 1.2 mL of phosphate buffered saline (PBS) was extracted to resuspend the cells obtained by centrifugation, 30 μL of cell suspension was taken and added to a cell smearing machine with a single-hole funnel, centrifuged at 1000 rpm for 5 min to make the cells spin onto a glass slide, and then naturally air-dried; 0.2-0.5 mL of A solution was added dropwise onto the glass slide, and dyed for 1 min; 1-2 mL of B solution was added dropwise onto the A solution, and the two solutions were mixed thoroughly, dyed for 5 min, then gently washed with running water, naturally air-dried, and then mounted, observed under a microscope, and counted the cells.

[0208] 8. Statistical analysis

[0209] Statistical analysis of clinical data was performed using SAS 9.4 software (SAS Institute, Cary, NC) and SPSS 27.0 software. Categorical variables were summarized using percentages and compared with chi-square or exact probability tests. Continuous variables were expressed as mean ± standard deviation (SD) and compared with independent t-tests or Wilcoxon rank-sum tests. Paired t-tests were used to test differences in mean values within groups. Multiple comparisons were compared using one-way analysis of variance (ANOVA) and Kruskal-Wallis tests. In all analyses, P less than 0.05 was considered significant. In addition, all graphics were created using GraphPad Prism (9.0) software.

[0210] II. Results

[0211] 1. LHQK showed clinical efficacy in patients with severe pneumonia after 7 days of treatment

[0212] In the ongoing clinical study, a total of 80 patients were enrolled, of which 30 (37.5%) were female and 50 (62.5%) were male. The average age of the patients was 68.4 ± 10.11 years. The average duration of illness before the study was 22.5 hours, and 45%, 35.0%, and 20% of the patients had comorbidities such as hypertension, new coronavirus infection, and stroke, respectively. The 80 patients were randomly divided into the LHQK group, including LHQK-L (n = 20) and LHQK-H (n = 20), or the basic treatment group (n = 40). The baseline characteristics of the LHQK group and the basic treatment group were comparable. No statistically significant differences were observed between the two groups in terms of demographic characteristics, disease duration, vital signs, and medical history.

[0213] The clinical efficacy of LHQK tablets was evaluated by assessing various parameters, including the value and change rate of APACHE II score, improvement in chest X-ray or CT, arterial partial pressure of oxygen (PaO2), arterial oxygen saturation (SaO2), oxygenation index (P / F), and hospitalization time after 7 days of treatment. At the baseline, there were no significant differences in the indicators of patients with severe pneumonia between the LHQK group and the basic treatment group. On the 8th day, the basic treatment group showed no significant improvement in PaO2, SaO2, P / F ratio, lung inflammation, or hospitalization time reduction. In contrast, compared with the basic treatment group, taking LHQK led to a significant improvement in chest X-ray or CT scan and inflammatory status in APACHE II score. In addition, LHQK showed increased P / F, PaO2, and SaO2 levels, increased oxygen uptake in patients with severe pneumonia, and improved ventilation function, with a shorter hospitalization time (Table 1).

[0214] Table 1 Average change between baseline and end of treatment

[0215]

[0216] In summary, LHQK can reduce the severity of severe pneumonia in patients and shorten hospitalization time. In addition, pathological study results show that LHQK can repair the airway epithelial barrier in patients, reduce sputum viscosity, reduce airway MUC5AC protein levels, repair airway epithelial cilia cell defects, and reduce the number of BALF inflammatory cells, which collectively contribute to the therapeutic effect on severe pneumonia.

[0217] Example 2 Protective effect of LHQK on lipopolysaccharide-induced severe pneumonia model mice

[0218] I. Materials and Methods

[0219] 1. Experimental materials

[0220] 1.1 Experimental animals

[0221] 6-8 weeks male SPF level BALB / c mice (body weight 19-21 g) (animal license number NO. 110011220110469445) were purchased from Beijing Vantianlihua Experimental Animal Technology Co., Ltd.

License number SCXK (Jing) 2021-0006

SYXK (Ji) 2020-003

[0222] 1.2 Experimental drugs and reagents

[0223] LHQK tablets Shijiazhuang Lingfeng Pharmaceutical Co., Ltd.

[0224] Lipopolysaccharide Beijing Solabio Technology Co., Ltd.

[0225] Common feed Beijing Kaoshe Li Feed Co., Ltd.

[0226] Corn cob bedding Beijing Kaoshe Li Feed Co., Ltd.

[0227] Lipopolysaccharide Beijing Solabio Technology Co., Ltd.

[0228] Isoflurane Hong Kong Youcheng Biological Technology Co., Ltd.

[0229] Sodium pentobarbital Merck Company, USA

[0230] 4% paraformaldehyde solution Wuhan Saiver Biological Technology Co., Ltd.

[0231] Collagenase type 2 Worthington Biochemical Company, USA

[0232] Elastase Suspension Worthington Biochemical Company, USA

[0233] Deoxyribonuelease I Beijing Solabio Biological Technology Co., Ltd.

[0234] Dispase II Beijing Solabio Biological Technology Co., Ltd.

[0235] 70 μm cell mesh screen Beijing Shuohuabiaoao Biological Technology Co., Ltd.

[0236] DMEM medium Gibco Company, USA

[0237] Fetal Bovine Serum Gibco, USA

[0238] Phosphate Buffer Gibco, USA

[0239] Cell Staining Buffer Biyun Tian Biotech Co., Ltd.

[0240] BSA Stock Solution Meitean Biotech Co., Ltd.

[0241] Rinsing Solution Meitean Biotech Co., Ltd.

[0242] Serum-free Cell Freezing Medium Saivon Biotech Co., Ltd.

[0243] Neutral Gum Mounting Medium Solabio Biotech Co., Ltd.

[0244] CD45 MicroBeads Meitean Biotech Co., Ltd.

[0245] Antibody Diluent Yakeyin Biotech Co., Ltd.

[0246] CD4 Antibody Abeam, USA

[0247] CD31 Antibody CST, USA

[0248] CD8 Antibody CST, USA

[0249] CD19 Antibody CST, USA

[0250] Ly6G Antibody CST, USA

[0251] F4 / 80 Antibody CST, USA

[0252] Goat Anti-Rabbit IgG H&L 594) Abeam, USA EDTA Solution Solabio Biotech Co., Ltd. Multicolor Immunofluorescence Immunohistochemistry Staining Kit Bainuo Panorama Co., Ltd. Tyramide Signal Amplification Staining Kit Bainuo Panorama Co., Ltd. Multicolor Immunofluorescence Immunohistochemistry Staining Kit Yunkunlon Technology Co., Ltd. DAPI SIGMA, USA

[0253] TBS Saivell Biotech Co., Ltd.

[0254] 20% Tween Solabio Biotech Co., Ltd.

[0255] 1.3 Main Instruments Electronic Balance Shanghai Yueping Scientific Instrument Co., Ltd.

[0256] Precise electronic balance, Germany, KERN company

[0257] General type small animal anesthesia machine, Shenzhen Ruivode Life Science and Technology Co., Ltd.

[0258] Tissue embedding machine, Japan, Sahura company

[0259] Tissue section staining machine, Japan, Sahura company

[0260] RS-28 type high-speed low-temperature centrifuge, Germany, Heraeus company

[0261] High-speed refrigerated centrifuge, Wuhan Gene Technology Co., Ltd.

[0262] Large high-speed low-temperature centrifuge, Beckman company, USA

[0263] DGX-9073 air blast drying oven, Shanghai Fuma Experimental Equipment Co., Ltd.

[0264] Multifunctional enzyme label instrument, BioTek company, USA

[0265] Ultralow temperature refrigerator, Germany, Heraeus company

[0266] XW-80 vortex mixer, Haimen Qishilainbail Instrument Manufacturing Company

[0267] 2. Experimental method

[0268] 2.1 Preparation of main drugs

[0269] 2.1.1 Selection and preparation of drug dose

[0270] LHQK tablets (batch number SYB2112001) were ground into ultrafine powder, produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd., with a specification of 20 g / bag and a crude drug content of 4.78 g / g. The adult clinical dosage of LHQK tablets is 22.2 g of crude drug per day, and according to the dose conversion relationship between humans and mice, the equivalent dose of mice is about 10 times that of humans (based on the body weight of 60 kg for an adult). Therefore, the equivalent crude drug amount of each mouse in the low-dose group of LHQK tablets should be 10 x 22.2 g / 60 kg = 3.7 g / kg / day, and that in the high-dose group should be 7.4 g / kg / day. 0.78 g and 1.6 g of LHQK tablet powder were taken into a mortar, respectively, 3 ml of distilled water was added, and they were ground thoroughly, and then made up to 10 ml and suspended thoroughly. According to the body weight of mice, they were administered intragastrically at a dose of 10 ml / kg, and stored in a 4°C refrigerator for standby.

[0271] 2.1.2 Preparation of Lipopolysaccharides (LPS) modeling drugs

[0272] LPS purity ≥98%, produced by Beijing Solabio Technology Co., Ltd., resuspended in 1 mL of normal saline according to the ratio of 1 mg powder, placed in a vortex instrument and vortexed until the powder was completely dissolved, to obtain a 1 mg / mL solution. Stored in a 4°C refrigerator, stable for about a month.

[0273] 2.2 Animal experiment grouping, model establishment, and model establishment standard

[0274] 2.2.1 Animal experiment grouping

[0275] A total of 40 BALB / c mice were randomly divided into a control group (Control), a model group (Model), a LHQK tablet low-dose group (LHQK-L), and a LHQK tablet high-dose group (LHQK-H).

[0276] 2.2.2 Model establishment

[0277] LPS intraperitoneal injection: LPS intraperitoneal injection was performed at a dose of 12 mg / kg on the first day of experimental modeling. LPS airway instillation: isoflurane gas anesthesia was used on the sixth day of experimental modeling, and airway instillation was performed at a dose of 4 mg / kg. The anesthetized mice were fixed in a supine position with the head higher than the tail on the operation table, the glottis was fully exposed, a 1 mL syringe was used to extract the required LPS solution, the needle was inserted into the mouse airway under light source irradiation, after successful intubation, the solution was quickly injected into the airway, the mouse fixation plate was quickly erected, and the mouse lungs were gently rubbed and shaken left and right, so that the LPS solution could be evenly distributed in the lungs. Sampling was performed on the seventh day of modeling. LHQK gavage was started four days before LPS intraperitoneal injection and continued until 24 hours after LPS tracheal instillation.

[0278] 2.3 Diagnosis standard

[0279] In view of the current modeling standards, the "China Emergency Critical Pneumonia Clinical Practice Expert Consensus" published by the Emergency Physician Branch of the China Medical Association in 2016 and the "China Adult Community Acquired Pneumonia Diagnosis and Treatment Guidelines" published by the Respiratory Disease Branch of the Chinese Medical Association in 2016 were referred to, and the pre-experiment results of different modeling conditions were combined to determine the diagnosis standard for the success of the mouse severe pneumonia model in this study: (1) reaction is slow or even consciousness is impaired, food and water intake is reduced, etc.; (2) respiratory rate increases, obvious double thoracic movement is visible; (3) body weight decreases significantly; (4) blood and double lungs show typical inflammatory manifestations: inflammatory cell infiltration, bleeding, and significant increase in inflammatory factor levels in lung tissue, etc.

[0280] 2.4 Index detection

[0281] 2.4.1 Multi-factor assay

[0282] The protein levels of Interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-12, IL-13, IL-17, C-X-C motif chemokine ligand 1 (CXCL1), Tumor Necrosis Factor alpha (TNF-α), Monocyte chemotactic protein-1 (MCP-1), Forkhead box protein P3 (FOXP3), Granulocyte colony-stimulating factor (GCSF), Macrophage-stimulating factor (MCSF), and Matrix Metalloproteinase-9 (MMP9) in lung tissue supernatant were detected using a multi-factor assay kit. The supernatant was first diluted 10-fold, and 100 μL of the standard or sample and 10 μL of magnetic beads were added to a 96-well plate, which was incubated at 37°C on a plate shaker for 90 min. The magnetic beads were then washed, 100 μL of detection solution A was added, and the plate was incubated at 37°C on a plate shaker for 60 min. Then, 100 μL of detection solution B was added after the plate was washed three times with a magnet, and the plate was incubated at 37°C on a plate shaker for 30 min. The plate was washed with a magnet again three times, and finally, 100 μL of sheath fluid was added, and the plate was vortexed and shaken for 2 min for reading.

[0283] 2.4.2 Multi-color immunofluorescence

[0284] Multi-color immunofluorescence staining was performed using a PANO 7-plex kit (Panovue, 0004100100). After the mouse lung tissue sections were deparaffinized and the antigen was repaired by microwave and cooled to room temperature, blocking solution was added for blocking, and endothelial cells-CD31 (CST, CST77699, 1:200), CD4 + T cells-CD4 (Abeam, ab183685, 1:200), CD8 +T cell-CD8 (CST, CST98941, 1:100), B cell-CD19 (CST, CST90176, 1:100), neutrophil-Ly6G (CST, CST87048, 1:100) and macrophage-F4 / 80 (CST, CST70076, 1:100) were incubated with 6 different primary antibodies for 1 h at room temperature, and then washed with TBST for 3 min. Then, HRP-conjugated secondary antibodies were added and incubated for 10 min, and then washed with TBST for 3 min. TSA (1:100) was added for fluorescent staining to amplify the signal, and then washed with TBST for 3 min. The sections were subjected to microwave repair, and then cooled to room temperature and washed with TBST for 3 min. The nuclei were stained with DAPI. Finally, the images were analyzed using a microscope.

[0285] 2.4.3 Olink assay

[0286] The extracted lung tissue supernatant was subjected to protein microarray, and 92 protein biomarkers were analyzed. The oligonucleotide-labeled antibody probe binds to its target protein, and if the two probes are very close, the oligonucleotide will hybridize in pairs. PCR amplification is performed immediately after hybridization and extension, and finally the amplicon is quantified by microfluidic qPCR, and the data is expressed as standardized protein expression values.

[0287] 2.4.4 Mass cytometry (Cytometry Time Of Flight, CyTOF)

[0288] The freshly isolated lung tissue was cut and transferred to 3 mL enzyme digestion solution, which contained 2 mg Collagenase type 2, 0.04 mg Elastase Suspension, 5 U Deoxyribonuelease I and 25 μL Dispase II per 1 mL enzyme digestion solution, and incubated at 37°C on a shaker for 20 min. Then, 10-fold volume of DMEM containing 10% FBS was added to terminate digestion, and the single cell suspension was blown 10 times by a needle syringe, and then filtered through 100 μm, 70 μm and 40 μm filters, respectively. The cell suspension was centrifuged at 400 g and 4°C for 5 min. 10 μL CD45 MicroBeads and 90 μL buffer at 4°C for 1 hour, and then sorted for CD45 cells using a manual + magnetic separator to obtain fresh CD45 +Cells; 500 μL of cisplatin was added and incubated at room temperature for 2 min, and 1.5 mL of Cell Staining Buffer was added to terminate the labeling, and centrifuged at 300 g for 5 min at room temperature; then 1.5 mL of 1.6% paraformaldehyde was added, repeatedly blown and incubated at room temperature for 10 min to reduce cell dimerization, and finally labeled with different antibodies, using metal tags instead of traditional fluorescent flow-through fluorescent tags, and using an inductively coupled plasma tandem TOF mass detector to perform high-speed, full-spectrum, high-resolution quantitative analysis of metal isotopes.

[0289] 3 Statistical processing

[0290] SPSS 27.0 statistical analysis software was used for data analysis, and measurement data was expressed as median or mean ± standard deviation (x ± s). Multiple samples were first subjected to normality test and variance homogeneity test, if the data were normal and variance homogeneity, one-way ANOVA was used; if the data were normal distribution, but the variance was not homogeneous, Dunnetts T3 method was used for comparison analysis; if the data were not normal distribution, non-parametric test was used for analysis (Kruskal-Wallis test); P<0.05 was considered to have statistical significance. All graphics were created using GraphPad Prism (9.0) software.

[0291] II. Results

[0292] 1. LHQK improves abnormal cytokine levels in the lungs of mice with severe pneumonia

[0293] The multi-factor results show (Table 2) that the levels of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12, IL-13, IL-17, CXCL1, TNF-α, MCP-1, FOXP3, GCSF, MCSF, and MMP9 inflammatory factors in the lung tissue supernatant of the Control group mice were lowly expressed; compared with the Control group, the levels of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12, IL-13, IL-17, CXCL1, TNF-α, MCP1, FOXP3, GCSF, MCSF, and MMP9 inflammatory factors in the lung tissue supernatant of the Model group were significantly increased (P<0.001), but compared with the Model group, the levels of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12, IL-13, IL-17, CXCL1, TNF-α, MCP1, FOXP3, GCSF, MCSF, and MMP9 were inhibited in the LHQK-L and LHQK-H groups (P<0.001).

[0294] Table 2 Factor protein expression levels in lung tissues of each group detected by multifactor kit

[0295]

[0296] Olink's volcano map results show that ( Figure 1 ), compared with the Control group, 43 proteins including CXCL1, IL-1α, CC motif chemokine ligand 3 (CCL3), CSF2, IL-1β, CCL20, CCL5, TNF, CCL2, IL-23r, CXCL9, IL-6, and IL-10 were significantly upregulated in the lung tissue supernatant of the Model group, while 12 proteins including nicotinamide adenine dinucleotide (Molecular properties and regulation of NAD+kinase, NADK), Activin A receptor like type 1 (ACVRL1), and mitogen-activated protein kinase kinase 6 (MAP2K6) were significantly downregulated. At the same time, compared with the Model group, two proteins were found to be upregulated in the lung homogenate of the LHQK group (i.e., LHQK-L group, the same below), while 28 proteins including phospholipase A2 group IVA Gene, PLA2G4A, fibronectin leucine rich transmembrane protein 2Gene, FLRT2, cytopathic effect (CPE), calcium activated nucleotidase 1Gene, CANT1, TNF receptor superfamily member 11bGene, TNFRSF11B), IL-10, CCL5, IL-23r, CCL2, etc. were significantly downregulated.

[0297] The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that ( Figure 2), Control, Model and LHQK groups were significantly changed in cytokine activity, inflammatory response, extracellular space, extracellular components and inflammatory factors of immune response. In addition, the top 10 pathways highlighted the involvement of signaling pathways related to cytokine-cytokine receptor interaction and inflammatory signaling pathways mediated by inflammatory factors, including TNF, IL-17, mitogen-activated protein kinases (MAPK), Toll-like receptor (TLR) and T helper cell 17 (Th17).

[0298] Trend analysis of the gradient-sorted expression data was performed by Short Time-series Expression Miner (STEM), and the trend analysis results showed that Figure 3 According to the changes in expression between Control, Model and LHQK groups, the differential proteins were divided into different trends (P<0.01). Two groups of immune proteins were lowly expressed in the Control group, significantly up-regulated in the Model group, and slightly or significantly down-regulated in the LHQK group (P<0.05). The heatmap results showed that inflammatory factors, including chemokines CXCL1, CCL2, CCL3, CCL5, CXCL9 and CCL20, and cytokines IL-5, IL-6, IL-α, IL-1β, IL-10, IL-17A and IL-17F, etc. were up-regulated in the Model group and down-regulated in the LHQK group, which were related to these two trends.

[0299] The results of multi-factor and Olink showed that there was an abnormal immune response in the mice with severe pneumonia, which was characterized by the simultaneous occurrence of high inflammatory response and immune suppression. LHQK treatment attenuated the up-regulation of pro-inflammatory cytokines such as IL6, IL-1β, TNF-α and the up-regulation of anti-inflammatory cytokines such as IL-4, IL10, FOXP3, thereby restoring the balance of the dysregulated cytokine environment. The results of previous studies showed that for other airway inflammation (such as acute bronchitis), the pathological changes were high expression of low inflammatory factors such as IL-6, IL-8, TNF-α, and low expression of anti-inflammatory factors IL-10, while in the severe pneumonia model, both inflammatory factors and anti-inflammatory factors were lowly expressed, which indicated that the mechanisms of severe pneumonia and general airway inflammation were essentially different.

[0300] 2. LHQK can reverse the immune disorder caused by macrophage activation and lymphocyte depletion in mice with severe pneumonia

[0301] Multicolor immunofluorescence results showed (Table 3) that the number of macrophages and neutrophils in the Model group was significantly increased (P<0.001) compared with the Control group, while the number of CD4 + T cells, CD8 + T cells and B + cells was significantly reduced (P<0.001); compared with the Model group, the proportion of macrophages and neutrophils in the LHQK group was significantly reduced (P<0.001, P<0.05), while the number of CD4 + T cells, CD8 + T cells and B cells was significantly increased (P<0.01, P<0.001, P<0.001).

[0302] Table 3. The positive cell rate in lung tissue of each group was determined by multicolor immunofluorescence method (n=3, )

[0303]

[0304] Note: ***p<0.001, compared with the Control group; #p<0.05; ##p<0.01, ###p<0.001, compared with the Model group.

[0305] The T-distributed stochastic neighbor embedding (T-SNE) dimensionality reduction map and heat map results of CyTOF technology showed that (Fig. 3), Figure 4 CD45 cells in the lung tissue of mice were co-defined by eight classical typing markers into seven major immune cells, including B cells, CD4+T cells, CD8+T cells, macrophages, granulocytes, NK cells and NK T cells.

[0306] The box plot results showed that (Fig. 4), + compared with the Control group, the proportion of B cells, CD4 + T cells and CD8 + T cells in the Model group was significantly reduced (P<0.05). While the proportion of macrophages was significantly increased (P<0.05). Notably, compared with the Model group, the proportion of B cells, CD4 + T cells and CD8 + T cells in the LHQK group was significantly increased (P<0.05), while the proportion of macrophages was significantly reduced (P<0.05), and the proportion of granulocytes had a decreasing trend (P=0.05).

[0307] Subsequently, 21 cell subgroups with different phenotypes were identified using 36 cell surface molecular markers (Fig. 5), Figure 6 including 4 types of CD4+ T cells, Class 3 CD8 + T cells, Class 4 macrophages, Class 2 B cells, Class 2 granulocytes, Class 1 NK cells, Class 2 NK T cells, and Class 3 undefined cells.

[0308] When using a combination of CD45, CD3e, CD4, TCRy5, TCRp, CD8, TNFa, CD69, CD103 cell surface markers, four classes of CD4 + T cells Figure 7 ) were revealed, including two clusters of T Helper 1 cells (Th1) (CD45 + CD3e + TCRy5 - TCRp + CD4 + CD8 - TNFa + ) subpopulations, tissue resident memory T cells (Trm) (CD3 + CD4 + CD69 + CD103 + ), and Regulatory T cells (Treg) (CD45 + CD3e + TCRy5 - TCRp + CD4 + CD8 - IL - 10 + ) were revealed. Among these cell subpopulations, the proportion of CD4hi, CD3eTh1 cells was significantly reduced (P<0.05), the proportion of CD4lo, CD3eTh1 cells was reduced, and the proportion of Treg cells was significantly increased (P<0.05) in the Model group compared with the Control group; compared with the Model group, the reduction of two clusters of Th1 cells was alleviated in the LHQK group, and the proportion of Treg cells was significantly reduced (P<0.05).

[0309] When using a combination of CD3, CD44, CD62, CD127, CD45, CD3e, TCRy5, TCRp, CD4, CD8a, CD69 cell surface markers, the presence of three classes of CD8 + T cells Figure 8 ) was revealed, including Effector memory T cells (Tem) (CD3 +CD44hi CD62Llo CD127 + ) and two clusters of activated T cells (Activated T) (CD45 + CD3e + TCRy5 - TCRp + CD4 - CD8a + CD69 + ) were significantly reduced in the Model group compared to the Control group (P<0.05); while the reduction of activated T cells in the LHQK group was improved compared to the Model group (P<0.05).

[0310] When using the cell surface marker combination of CD45, CD3e, CD19, CD14, CD49b and Gr1, two types of granulocytes were revealed (CD45 Figure 9 ), including mast cells (CD45 + CD3e - CD19 - CD14lo IL-4 + CD49b + ) and neutrophils (Gr1+). The Model group showed a higher proportion of neutrophils compared to the Control group; while the proportion of neutrophils in the LHQK group was reduced compared to the Model group.

[0311] When using the cell surface marker combination of CD11b, CD11c, F4 / 80, CD86 and Siglec-F, the presence of four types of macrophages was revealed (CD11b Figure 10 ). They are alveolar macrophages (AM) (CD11b - CD11c + Siglec-F + ), two types of interstitial macrophages (IM) (Siglec-F - CD11c - CD11b - F4 / 80 + ) and alternatively activated macrophages (M2) (CD11b + F4 / 80 + CD206 +). Compared with the Control group, the proportion of AMs in the Model group was significantly reduced (P<0.05), and the proportion of two IMs cells tended to increase; and compared with the Model group, the proportion of AMs in the LHQK group was significantly increased (P<0.05), and the proportion of IMs cells was reduced.

[0312] The results of polychromatic immunofluorescence and CyTOF show that LHQK can alleviate the activation of macrophages and the depletion of lymphocytes (B cells, CD4 + T cells, CD8 + T cells) in mice with severe pneumonia.

[0313] In summary, the drug of the present application can reduce pro-inflammatory and anti-inflammatory factors to inhibit high inflammatory response, and can alleviate immune depletion by adjusting macrophages and lymph T cells, thereby correcting the immune disorder caused by severe pneumonia, which is an important factor for treating severe pneumonia, and provides a basis for the clinical application of the drug for treating severe pneumonia.

[0314] Test Example 3 Protective effect of LHQK on human pulmonary microvascular endothelial cells (HPMECs)

[0315] 1 Experimental method

[0316] 1.1 HPMECs culture

[0317] (1) Cell recovery and culture

[0318] The cell cryopreservation tube in the liquid nitrogen tank was quickly transferred to a 37℃ water bath pot, and after it was dissolved, it was centrifuged at 1000 rpm for 3-4 min, and the supernatant was slowly removed in the clean bench, 1 mL of ECM complete medium was added to the tube, and the cells were gently blown evenly, then it was inoculated in a 10 cm culture dish containing 10 mL of preheated ECM complete medium, and it was gently mixed in an "8" shape, then it was placed in a 37℃, 5% CO2 cell incubator for culture.

[0319] (2) Cell passage

[0320] When the cell density was 80-90%, the passage was started. The old culture medium was discarded, and 4 mL of PBS was used for 2 times of washing. 1 mL of trypsin was added, and then it was placed in the incubator for 1-3 min, when the cells were round and floated, 1 mL of culture medium was immediately added to terminate digestion, the bottom of the dish was inclined, and the non-digested cells on the bottom of the dish were gently blown from the inclined upper to the lower to ensure complete detachment, and then they were sucked into a 15 mL centrifuge tube, centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the culture medium was added and mixed evenly, and the passage was carried out at a ratio of 1:2, and the cells within 6 generations were used for subsequent experiments.

[0321] (3) Cell counting

[0322] Cell digestion and centrifugation steps as before, light blow mixed cell suspension, try to avoid bubbles, pipette 10 μl cell suspension to make it fully distributed in the cell counting plate, cell counter counting.

[0323] (4) cell cryopreservation

[0324] Cell digestion and centrifugation, discard the supernatant, add cell freezing solution into the centrifuge tube, blow the cells evenly, transfer to the cryopreservation tube, seal the tube with sealing film, put it into the cryopreservation box, and then put it into the liquid nitrogen tank for long-term preservation.

[0325] 1.2 cell drug treatment, model establishment and grouping

[0326] First, pre-experiment, through CCK-8 experiment to select the best intervention concentration of LPS and LHQK, cells were seeded in 96-well plates at a density of 1×10 5 Cells / mL, and placed in a cell incubator for 24-48 h. When the confluence of monolayer cells was 80%-90%, the old culture medium in the culture dish was removed, and PBS was used to wash twice. Different concentrations of LPS (1, 10, 100 μg / ml) were added to each well with 100 μl, and incubated for 6, 12, and 24 h, respectively. The best intervention concentration and time of LPS were determined by CCK-8 detection of cell viability. Next, different concentrations of LHQK (1, 50, 125, 250, 500 μg / mL) were diluted and incubated with cells in a cell incubator for 24 h. The non-toxic drug concentration of LHQK was determined by CCK-8 results. Finally, the cells were pretreated with non-toxic drug concentration of LHQK (1, 50, 125, 250 μg / mL) for 2 h, and then induced acute cell injury model with LPS (10 μg / ml). After 24 h of treatment, the cell survival status was evaluated by CCK-8, and the best treatment concentration of LHQK was selected. The subsequent experiment was divided into groups: HPMECs (4-6 passages) were divided into 4 groups: normal group (Control), model group (Model), LHQK low dose group (LHQK-L), and LHQK high dose group (LHQK-H). The Model group was stimulated with LPS (10 μg / mL) for 24 h to establish a barrier injury model. The LHQK-L and LHQK-H groups were pretreated with LHQK 125 μg / mL and LHQK 250 μg / mL, respectively, for 2 h before being incubated with LPS for 24 h. The specific grouping is as follows:

[0327] ① Control group: HPMECs were not treated;

[0328] ② Model group: HPMECs were treated with LPS (10 μg / mL) for 24 h;

[0329] 3. LPS+LHQK-L: LPS (10 μg / mL) + LHQK (125 μg / mL) for 24 h;

[0330] 4. LPS+LHQK-H: LPS (10 μg / mL) + LHQK (250 μg / mL) for 24 h.

[0331] 2. Experimental results

[0332] 2.1 Effect of LHQK on the activity of HPMECs

[0333] CCK-8 results showed that after stimulating HPMECs with LPS (1, 10, 100 μg / mL) for 6 h, no significant difference in cell activity was detected compared with the control group. After stimulating HPMECs with LPS (10 μg / mL) for 12 h, a decrease in cell activity was detected. After stimulating HPMECs with LPS (10 μg / mL) for 24 h, a significant decrease in cell activity was detected. According to the IC50 calculation formula, the concentration at half the effective amount was 10 μg / mL and the time was 24 h. Therefore, 10 μg / mL of LPS for 24 h was selected as the best stimulation time and concentration (Table 4). Next, CCK-8 detection of LHQK drug concentration results showed that, except for LHQK 500 μg / mL concentration which had damage to cells (P < 0.01), the rest of the concentrations had no damage to cells (Table 5). Finally, after pretreating HPMECs with non-toxic drug concentrations (1, 50, 125, 250 μg / mL) for 2 h, LPS (10 μg / mL) was added to induce an acute cell damage model. CCK-8 detection of cell activity results showed that LHQK at 50, 125 and 250 μg / mL could increase cell activity (P < 0.01). Therefore, 125 μg / mL and 250 μg / mL were selected as the best treatment doses of LHQK low dose and LHQK high dose for subsequent experiments (Table 6).

[0334] Table 4 Effect of LPS on the activity of HPMECs (n = 6, x ± s)

[0335]

[0336] Note: ** P < 0.01 vs. the Control group;

[0337] Table 5 Effect of LHQK on the activity of HPMECs (n = 6, x ± s)

[0338]

[0339] Note: ** P<0.01 vs. the Control group.

[0340] Table 6 Effect of LHQK on the viability of HPMECs exposed to LPS (n=6, )

[0341]

[0342]

[0343] Note: ** P<0.01 vs. the Control group; ## P<0.01 vs. the Model group.

[0344] 2.2 Effect of LHQK on the death of HPMECs

[0345] To further determine whether LHQK can protect HPMECs from LPS-induced endothelial cell death, Calcein AM / PI staining was used to detect the death of HPMECs. The staining results showed that compared with the Control group, LPS stimulation led to a significant increase in the number of dead cells in HPMECs (P<0.01); compared with the Model group, the number of dead cells in the LHQK group was significantly reduced (P<0.01) (Table 7). The effect of LHQK on the death of HPMECs was further verified by western blot to detect the expression of pro-apoptotic protein Bax, and the results showed that compared with the Control group, the expression of Bax in the Model group was significantly increased (P<0.05); compared with the Model group, the expression of Bax in the LHQK group was significantly reversed (P<0.05) (Table 8).

[0346] Table 7 Death rate of HPMECs in each group (n=4, )

[0347]

[0348] Note: ** P<0.01 vs. the Control group; ## P<0.01 vs. the Model group.

[0349] Table 8 Western Blot method for detecting the expression of pro-apoptotic protein Bax in HPMECs in each group (n=3, )

[0350]

[0351]

[0352] Note: ** P<0.01 vs. the Control group; ## P<0.01 vs. the Model group.

[0353] 3. Effect of LHQK on barrier dysfunction of HPMECs

[0354] The results of TEER value experiment showed that compared with the Control group, the TEER value of the Model group was significantly decreased (P<0.01); compared with the Model group, the TEER value of the LHQK-L and LHQK-H groups was increased to different degrees (P<0.01) (Table 9).

[0355] The results of immunofluorescence intensity of VE-cadherin showed that compared with the Control group, the expression of VE-cadherin protein in the Model group was significantly decreased (P<0.01); compared with the Model group, the expression of VE-cadherin protein in the LHQK-L and LHQK-H groups was increased (P<0.01) (Table 10).

[0356] The results of Western Blot detection of VE-cadherin and Occludin between cells in each group showed that compared with the Control group, the expression of VE-cadherin and Occludin protein in the Model group was significantly decreased (P<0.05, P<0.01); compared with the Model group, the expression of VE-cadherin and Occludin protein in the LHQK-L and LHQK-H groups was increased (P<0.05, P<0.01) (Table 11).

[0357] Table 9 Changes in TEER value of HPMECs in each group (n=3, )

[0358]

[0359] Note: ** P<0.01 vs. the Control group; ## P<0.01 vs. the Model group.

[0360] Table 10 Immunofluorescence intensity of VE-cadherin protein in HPMECs in each group (n=3, )

[0361]

[0362] Note: ** P<0.01 vs. the Control group; ## P<0.01 vs. the Model group.

[0363] Table 11 Western Blot detection of the expression of VE-cadherin, Occludin in each group of HPMECs (n=3,

[0364]

[0365] Note: ** P<0.01 vs. the Control group; # P<0.05, ## P<0.01 vs. the Model group.

[0366] From the above experimental results, it can be seen that the drug of the present application can significantly improve the viability of HPMECs, reduce the death of HPMECs, protect the barrier function of HPMECs, and can effectively prevent the progression of lung inflammation or injury into severe pneumonia.

[0367] The drug effect of Examples 2-4 is slightly worse than that of Example 1, but also has a significant therapeutic or preventive effect on severe pneumonia.

[0368] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. Use of a Chinese medicine composition in the preparation of a medicament for treating or preventing severe pneumonia, characterized in that The Chinese medicine composition is prepared from the following raw materials in parts by weight: Ephedra 52-86; Gypsum 194-324; Forsythia suspensa 194-324; Scutellaria baicalensis 78-130; Morus alba bark 194-324; Bitter almond 78-130; Peucedanum peucedanum 78-130; Pinellia ternata 78-130; Tangerine peel 78-130; Fritillaria 78-130; Arctium lappa 78-130; Honeysuckle 78-130; Rhubarb 39-65; Platycodon grandiflorum 46-76; Licorice 39-65.

2. The use according to claim 1, characterized in that The Chinese medicine composition is prepared from the following raw materials in parts by weight: Ephedra 52; Gypsum 324; Forsythia suspensa 194; Scutellaria baicalensis 78; Morus alba bark 194; Bitter almond 130; Peucedanum peucedanum 78; Pinellia ternata 130; Tangerine peel 78; Fritillaria 78; Arctium lappa 130; Honeysuckle 130; Rhubarb 39; Platycodon grandiflorum 76; Licorice 65.

3. The use according to claim 1, characterized in that The Chinese medicine composition is prepared from the following raw materials in parts by weight: Ephedra 86; Gypsum 194; Forsythia suspensa 324; Scutellaria baicalensis 130; Morus alba bark 324; Bitter almond 78; Peucedanum peucedanum 130; Pinellia ternata 78; Tangerine peel 130; Fritillaria 130; Arctium lappa 78; Honeysuckle 78; Rhubarb 65; Platycodon grandiflorum 46; Licorice root 39.

4. The use according to claim 1, characterized in that The Chinese medicine composition is prepared from the following raw materials in parts by weight: Ephedra 69; Gypsum 259; Forsythia suspensa 259; Scutellaria baicalensis 104; Morus alba bark 259; Bitter almond 104; Peucedanum peucedanum 104; Pinellia ternata 104; Tangerine peel 104; Fritillaria 104; Arctium 104; Honeysuckle 104; Rhubarb 52; Platycodon grandiflorum 61; Licorice 52.

5. The use according to claim 1, characterized in that The Chinese medicine composition is prepared from the following raw materials in parts by weight: Ephedra 55; Gypsum 254; Forsythia suspensa 318; Scutellaria baicalensis 107; Morus alba bark 203; Bitter almond 107; Peucedanum peucedanum 82; Pinellia ternata 105; Tangerine peel 84; Fritillaria 125; Arctium lappa 122; Honeysuckle 113; Rhubarb 42; Platycodon grandiflorum 60; Licorice 50.

6. The use according to any one of claims 1 to 5, characterized in that The bitter almonds are fried bitter almonds, the fritillaria is fritillaria thunbergii, the honeysuckle is honeysuckle, and the pinellia is pinellia ternata.

7. The use according to any one of claims 1 to 5, characterized in that The active ingredients of the Chinese medicine composition are prepared by the following steps: A. Weigh Fritillaria thunbergii according to the formula ratio, crush into fine powder, and set aside; B. Weigh ephedra, forsythia, bitter almond, pinellia, burdock fruit, and rhubarb according to the formula ratio, add 40-70% ethanol and reflux extract twice, each time for 1-4 hours, add 8-10 times the amount for the first time and 6-9 times the amount for the second time, combine the extracts, filter, and recover ethanol from the filtrate under reduced pressure, and concentrate to a clear paste with a relative density of 1.14-1.16 measured at 60°C, and set aside; C. Take gypsum, white mulberry bark, peucedanum chinense, dried orange peel, honeysuckle, platycodon grandiflorum, and liquorice by prescription ratio, decoct twice with water, each 1-4 hour, add 9-11 times of amount for the first time, add 7-9 times of amount for the second time, the decoction merges, filters, and is concentrated to a clear paste with a relative density of 1.14-1.16 at 60 ℃ of thermal measurements, merge with the clear paste gained in step B, and standby; The fine powder obtained in step A and the combined clear paste obtained in step C together constitute the active ingredient of the pharmaceutical composition.

8. The use according to any one of claims 1 to 5, characterized in that The dosage form of the Chinese medicine composition is tablets, capsules, powders, granules, oral liquids, pills, tinctures, syrups, suppositories, gels, sprays or injections.

9. The use according to claim 8, characterized in that The tablet is prepared by the following steps: A. Weigh Fritillaria thunbergii according to the formula ratio, crush into fine powder, and set aside; B. Weigh ephedra, forsythia, bitter almond, pinellia, burdock fruit, and rhubarb according to the formula ratio, add 40-70% ethanol and reflux extract twice, each time for 1-4 hours, add 8-10 times the amount for the first time and 6-9 times the amount for the second time, combine the extracts, filter, and recover ethanol from the filtrate under reduced pressure, and concentrate to a clear paste with a relative density of 1.14-1.16 measured at 60°C, and set aside; C. Take gypsum, white mulberry bark, peucedanum chinense, dried orange peel, honeysuckle, platycodon grandiflorum, and liquorice by prescription ratio, decoct twice with water, each 1-4 hour, add 9-11 times of amount for the first time, add 7-9 times of amount for the second time, the decoction merges, filters, and is concentrated to a clear paste with a relative density of 1.14-1.16 at 60 ℃ of thermal measurements, merge with the clear paste gained in step B, and standby; D. Spray-dry the combined clear paste obtained in step C and collect the spray powder for later use; E. The spray powder obtained in step D and the fine powder obtained in step A are prepared into a soft material using ethanol as a binder, and the material is sieved and granulated; and tablets are obtained by conventional pharmaceutical methods.

10. The use according to any one of claims 1 to 5, characterized in that The Chinese medicine composition simultaneously regulates the immune excess and immunosuppression of severe pneumonia.

11. The use according to claim 10, characterized in that The Chinese medicine composition reduces the proportion of macrophages, increases B cells, CD4 + T cells and CD8 + The proportion of T cells.

12. The use according to any one of claims 1 to 5, characterized in that The Chinese medicine composition reduces the levels of pro-inflammatory factors and anti-inflammatory factors in severe pneumonia and maintains the homeostasis of the immune microenvironment.

13. The use according to claim 12, characterized in that The pro-inflammatory factors include IL6, IL-1β and TNF-α, and the anti-inflammatory factors include IL-4, IL10 and FOXP3.

14. The use according to any one of claims 1 to 5, characterized in that The application of the traditional Chinese medicine composition in preparing a medicine for protecting pulmonary microvessels.

15. The use according to claim 14, characterized in that The Chinese medicine composition improves the vitality of pulmonary microvascular endothelial cells; and / or reduces the death of pulmonary microvascular endothelial cells; and / or protects the barrier function of pulmonary microvascular endothelial cells.

16. The use according to claim 15, characterized in that The traditional Chinese medicine composition increases the transendothelial electrical resistance of pulmonary microvascular endothelial cells; and / or increases the expression of VE-cadherin and Occludin proteins in pulmonary microvascular endothelial cells.

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