Method for treating pneumonia and reo-ling granules used

Abstract

The present application relates to the method and the used heat lung clear granules of treating pneumonia, one aspect, it is related to the method such as investigating the effect of heat lung clear granules in treating pneumonia by determining serum TNF-alpha concentration, including the steps: mice are randomly divided, atomized modeling is used LPS, after modeling, heat lung clear granules are given by gavage, blood is taken from mice, serum is collected, at least one of the following items is determined: lung index, TNF-alpha, IL-1beta, LI-6, PGE2 content in serum, and the statistical result of the treatment effect of heat lung clear granules on mice with pneumonia is obtained according to the detection data.The present application also provides the use of heat lung clear granules as a pneumonia treatment drug.The present application shows that heat lung clear granules are a very good pneumonia treatment drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and relates to a traditional Chinese medicine preparation, particularly to a traditional Chinese medicine preparation called Relinqing Granules made from the medicinal herb Polygonum capitatum. It also relates to the preparation method of this traditional Chinese medicine preparation and its pharmaceutical uses, especially the use of Relinqing Granules in the preparation of drugs for treating pneumonia. The research results of this invention show that Relinqing Granules have a significant positive therapeutic effect on pneumonia. This invention confirms the efficacy of Relinqing Granules as a treatment for pneumonia through a study on the protective effect of LPS-induced acute pneumonia in mice. Background Technology

[0002] Pneumonia is an inflammation of the lungs and is a common respiratory disease. It can occur at any age, but young children, the elderly, and those with immunodeficiency or weakened immune systems are at higher risk. Severe cases can be fatal. According to the World Health Organization, pneumonia accounts for 75% of deaths from acute respiratory infections. Pneumonia is a very broad term, as any inflammation of the lungs can be caused by biological, physical, chemical, or other factors.

[0003] Regarding the classification of pneumonia, it can be divided into: nosocomial pneumonia and community-acquired pneumonia according to the source of infection. (2) Pneumonia can be caused by different pathogens. According to the etiology, pneumonia can be divided into: infectious pneumonia (according to the type of pathogen: including bacterial pneumonia, common bacteria include Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, etc.; viral pneumonia, common viruses include respiratory syncytial virus, influenza virus, parainfluenza virus, adenovirus, etc.; in addition, there are fungal pneumonia, mycoplasma pneumonia, chlamydia pneumonia, etc.), physical and chemical pneumonia (such as radiation pneumonia, aspiration pneumonia, lipoid pneumonia), and allergic pneumonia (such as allergic pneumonia and rheumatic pneumonia). (3) Due to the different pathogens and the body's reactivity, the location, extent of involvement and nature of the lesion of inflammation are often different. Inflammation occurring in the alveoli is called alveolar pneumonia (most pneumonias are alveolar), and inflammation involving the lung interstitium is called interstitial pneumonia. The lesion is called lobular pneumonia when it is limited to a lobule, segmental pneumonia when it involves a segment, and lobar pneumonia when it involves the entire or multiple lobes. There are also other types such as bronchiolitis. (4) According to the course of the disease: it is divided into acute pneumonia, persistent pneumonia and chronic pneumonia. Generally, the course of persistent pneumonia is 1 to 3 months. If it exceeds 3 months, it is considered chronic pneumonia. (5) According to the nature of the lesion, it can be divided into different types such as serous pneumonia, fibrinous pneumonia, purulent pneumonia, hemorrhagic pneumonia, caseous pneumonia, granulomatous pneumonia or organizing pneumonia. Common symptoms of pneumonia include: cough with yellow-green sputum; fever with chills; severe or stabbing chest pain, which is aggravated by deep breathing or coughing; rapid breathing; shortness of breath; high fever (body temperature at least 39.5°C).

[0004] Relinqing granules are a single-ingredient preparation made from Polygonum capitatum Buch.-Ham. exD. Don, a traditional Chinese medicine from Guizhou. It has the effects of clearing heat and purging fire, promoting diuresis and relieving strangury. It can be used to treat strangury caused by damp-heat in the lower jiao, with symptoms such as frequent urination, urgency and painful urination; urinary tract infection and pyelonephritis with the above symptoms. Clinically, it is often used to treat various symptoms caused by damp-heat accumulation in the lower jiao and poor qi transformation of the body. [Liang Bin, Zhang Liyan, Ran Maoxiong. Polygonum capitatum Buch. Chinese Miao medicine [M]. Beijing: China Traditional Chinese Medicine Press, 2014; Wang Chongyang, Pan Shu, Wu Yali, et al. Experimental study on the pharmacological effects of Relinqing granules [J]. Journal of Practical Traditional Chinese Internal Medicine, 2012, 26(03): 12-14; Nan Haifeng, Liu Jie, Wu Dan, et al. Clinical effect of Relinqing granules combined with conventional antibiotics in the treatment of gonorrhea and its influence on the expression of serum inflammatory mediators [J]. World Journal of Integrated Traditional and Western Medicine, 2021, 16(06): 1103-1107; National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China 2020 Edition (Part I) [S]. Beijing: China Medical Science and Technology Press, 2020.

[0005] Modern pharmacological studies have found that Relinqing granules can have a certain therapeutic effect on various inflammations such as gastritis, nephritis, and prostatitis, and inflammatory reactions often cause fever of varying degrees in the body. Traditional Chinese medicine believes that fever is the result of the struggle between pathogenic factors and the body's vital energy after the invasion of external pathogens. It was first recorded in the "Huangdi Neijing" and is called "body heat". It mostly belongs to the category of febrile diseases [Li Yingchao. Differentiation and treatment of fever in traditional Chinese medicine [J]. Journal of Traditional Chinese Medicine, 2010, 51(S1): 125-126; Dou Xiaoxin, Yang Yuying, Bu Zhichao, et al. Try to understand the 2019 novel coronavirus pneumonia from the perspective of traditional Chinese medicine [J]. Tianjin Journal of Traditional Chinese Medicine, 2020, 37(02): 137-140].

[0006] Lipopolysaccharide (LPS)-induced pneumonia is a commonly used pharmacological model for pneumonia research. LPS is a major component of endotoxins, originating from the outer membrane of Gram-negative bacterial cell walls. It is ubiquitous in polluted air, occupational dust (such as grain dust), and cigarette smoke. Occupational and environmental inhalation of certain concentrations of these substances can cause or exacerbate a series of clinical symptoms, such as asthma and bronchopneumonia. Bronchopneumonia induced by LPS can be cured with timely and effective treatment; otherwise, the disease course is prolonged, and repeated airway inflammation can develop into chronic bronchitis. Continued exposure to high concentrations of LPS will progressively worsen the condition, eventually leading to pulmonary heart disease. Studies have found that LPS induces the overexpression of various chemokines and inflammatory factors in vitro and in vivo. The main cytokines causing increased neutrophil aggregation in lung tissue are IL-1β and TNF-α.

[0007] Therefore, those skilled in the art expect to provide a method for determining the efficacy of Relinqing granules in treating pneumonia, and / or also expect to provide a Relinqing granule, and / or also expect to provide the use of Relinqing granules in the preparation of a medicament for treating pneumonia. Summary of the Invention

[0008] The purpose of this invention is to provide a method for determining the therapeutic effect of Relinqing granules on pneumonia, and / or to also provide Relinqing granules, and / or to also provide the use of Relinqing granules in the preparation of a medicament with therapeutic effects on pneumonia. This invention uses Relinqing granules as the research object to study its therapeutic effect on LPS-induced acute pneumonia in mice. It has been unexpectedly discovered that the therapeutic effect of Relinqing granules on pneumonia can be effectively determined using the method of this invention. This invention is based on this discovery.

[0009] Therefore, the first aspect of the present invention provides a method for determining the therapeutic effect of Relinqing granules on pneumonia, or the use of Relinqing granules in the preparation of a drug for treating pneumonia, or a method for examining the therapeutic effect of Relinqing granules on pneumonia by measuring serum TNF-α concentration, the method comprising the following steps:

[0010] BALB / c mice, half male and half female, were randomly divided into a blank control group, a model group, a Relinqing granule group, and a positive control group.

[0011] After the animals were acclimatized to the diet, LPS atomization was used to create the model.

[0012] The day after modeling, the Relinqing granules group and the positive control group were administered the drug by gavage, while the blank control group and the model group were administered an equal volume of physiological saline by gavage.

[0013] After administration, blood was collected from the eyeballs of mice. The blood was allowed to coagulate naturally at room temperature for 30 minutes, then centrifuged at 1000xg for about 15 minutes. The serum supernatant was collected and stored at 4°C for later testing (or frozen at -80°C).

[0014] Mice were euthanized by dislocation after blood collection, and bronchoalveolar lavage fluid and whole lung tissue were collected for analysis.

[0015] Determine at least one of the following items: after euthanizing mice by blood collection from their eyeballs, quickly and accurately weigh the entire lung and calculate the lung index;

[0016] Lung tissue specimens were dehydrated, trimmed, embedded, sectioned, stained, mounted, and examined under a microscope according to standard pathological examination procedures to observe pathological changes in the lung tissue.

[0017] The levels of TNF-α, IL-1β, LI-6, and PGE2 in the serum of mice in each group were detected using an ELISA kit.

[0018] Statistical results on the therapeutic effect of Relinqing granules on (acute) pneumonia mice were obtained based on the above test data.

[0019] According to the method of the first aspect of the present invention, the model is obtained by 3 consecutive days of modeling using LPS nebulization (20 mg / kg) after 7 days of animal acclimatization feeding (laboratory environment: 25±2℃, relative humidity 50±5%).

[0020] According to the method of the first aspect of the present invention, the Relinqing granules are divided into a high-dose group (6.24 g / kg), a medium-dose group (3.12 g / kg), and a low-dose group (1.56 g / kg).

[0021] According to the method of the first aspect of the present invention, the positive control group is orally administered dexamethasone acetate 0.878 / kg / d.

[0022] According to the method of the first aspect of the present invention, on the day following modeling, the high, medium and low dose groups of Relinqing granules and the positive control group were administered the drug by gavage, while the blank control group and the model group were administered an equal volume of physiological saline by gavage, once a day, for 7 consecutive days.

[0023] According to the method of the first aspect of the present invention, the procedures and conditions for processing lung tissue specimens according to the standard operating procedures for pathological examination, including dehydration, trimming, embedding, sectioning, staining, mounting, and microscopic examination, are as follows:

[0024] According to the method of the first aspect of the present invention, the dehydration conditions for lung tissue specimens according to the standard operating procedure for pathological examination are as follows: dehydration time: 75% alcohol 4h, 85% alcohol 2h, 95% alcohol 1h, 100% alcohol 0.5h, 100% alcohol 0.5h, 100% alcohol 0.5h, xylene 10min, xylene 10min, paraffin 1h, paraffin 2h, paraffin 3h;

[0025] According to the method of the first aspect of the present invention, the staining and mounting of lung tissue specimens according to the standard operating procedure for pathological examination are performed as follows: dewaxing the sections to water, staining with hematoxylin for 10-20 min, rinsing with tap water for 1-3 min, differentiating with hydrochloric acid alcohol for 5-10 s, rinsing with tap water for 1-3 min, immersing in warm water at 50°C or a weakly alkaline aqueous solution until blue appears, rinsing with tap water for 1-3 min, immersing in 85% alcohol for 3-5 min, staining with eosin for 3-5 min, rinsing with water for 3-5 s, dehydrating with graded alcohols, clearing with xylene, and mounting with neutral resin;

[0026] According to the method of the first aspect of the present invention, when examining lung tissue specimens under a microscope according to the standard operating procedure for pathological examination, an upright fluorescence microscope is used to acquire images of the slides. Each slide is first observed at 200x magnification to observe the entire tissue and gross lesions, and then the area to be observed is selected to acquire images at 400x magnification.

[0027] According to the method of the first aspect of the present invention, when detecting the levels of TNF-α, IL-1β, LI-6, and PGE2 in the serum of mice in each group by ELISA, the serum of rats in each group is taken, and the levels of TNF-α, IL-1β, LI-6, and PGE2 in the serum of mice in each group are detected according to the operation method of the ELISA kit instructions.

[0028] According to the method of the first aspect of the present invention, the experimental data obtained are expressed as mean ± standard deviation (s). The data are analyzed using statistical software (e.g., SPSS). When comparisons among multiple groups satisfy normal distribution and homogeneity of variance, one-way ANOVA is used for analysis. P A value <0.05 is considered statistically significant.

[0029] According to a method of a first aspect of the present invention, wherein the concentration of TNF-α is determined using a TNF-α ELISA kit, the method comprises the following steps:

[0030] a. Dilution of Standards: Take 6 small test tubes and number them sequentially. First, add 100µl of standard diluent to each test tube. Then, add 100µl of the original concentration standard to the first test tube and mix thoroughly. Next, add 100µl of the original concentration standard to the second test tube and mix thoroughly. Then, add 100µl of the original concentration standard to the third test tube and mix thoroughly. Then, add 100µl of the original concentration standard to the fourth test tube and mix thoroughly. Finally, add 100µl of the original concentration standard to the fifth test tube and mix thoroughly. Take 100µl of the solution and add it to the fifth test tube, then mix thoroughly. Take another 100µl of the solution from this tube and discard it. The sixth test tube serves as standard 0, and the concentrations after dilution are: 360 pg / ml, 180 pg / ml, 90 pg / ml, 45 pg / ml, 22.5 pg / ml, and 0 pg / ml. Set up standard wells on an enzyme-labeled plate, and add 50µl of different concentrations of standard solution sequentially, creating 5 parallel wells for each concentration.

[0031] b. Sample preparation and loading: Collect blood using blood collection tubes, allow the blood to coagulate naturally at room temperature for 30 minutes, centrifuge at 1000xg for approximately 15 minutes, collect the supernatant (serum), and mix the supernatant with the additional solution at a volume ratio of 9:1 to obtain the serum sample to be tested; set up sample wells and blank control wells (no sample or enzyme-labeled reagent is added to the blank control wells, and the remaining steps are the same as for the sample wells); add 40 μl of sample diluent to the sample wells on the enzyme-labeled plate, and then add 10 μl of the sample to be tested; when loading the sample, add the sample to the bottom of the well, avoiding contact with the well wall as much as possible, and gently shake to mix; the additional solution is an aqueous solution containing 1.25% glycine and 0.2% zinc chloride;

[0032] c. Incubation: Add 50 μl of enzyme-labeled reagent to each well, except for the blank control wells; seal the plate with sealing film and incubate at 37°C for 30 minutes;

[0033] d. Washing: Dilute the 30x concentrated washing solution with distilled water to obtain the washing solution; carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry;

[0034] d. Color development: Add 50 μl of color reagent A to each well, then add 50 μl of color reagent B, gently shake to mix, and develop color at 37°C in the dark for 10 minutes; then add 50 μl of stop solution to each well to stop the reaction, at which point the blue color will turn yellow.

[0035] e. Measurement and calculation: Within 15 minutes after adding the stop solution, zero the blank control well and measure the absorbance (OD) value of each well sequentially at a wavelength of 450 nm using an ELISA reader; calculate the linear regression equation of the standard curve using the concentration of the standard and the OD value, and calculate the sample concentration using the sample OD value.

[0036] According to the method of the first aspect of the present invention, the various materials used are conventional materials.

[0037] According to the method of the first aspect of the present invention, the ELISA kit used is as described in the examples.

[0038] According to the method of the first aspect of the present invention, the various materials used are conventional materials.

[0039] According to the method of the first aspect of the present invention, the ELISA kit used is as described in the examples.

[0040] Furthermore, a second aspect of the present invention relates to a heat-clearing granule, which is a granule made from Polygonum capitatum Buch.-Ham. ex D. Don.

[0041] According to the second aspect of the present invention, the hot-draining granules are prepared by the following method: 1250g of Polygonum hydropiper is taken, decocted twice with water for 1.5 hours each time, the decoction is filtered, the filtrates are combined, concentrated to an appropriate amount, filtered, spray-dried, mixed with an appropriate amount of soluble starch, made into granules, dried, and made into 500g, which is the final product.

[0042] Furthermore, a third aspect of the present invention relates to the use of the Relinqing granules described in any of the second aspects of the present invention in the preparation of a medicament for treating pneumonia.

[0043] Any technical feature of any aspect or embodiment of the present invention is equally applicable to any other embodiment or embodiment of any other aspect, provided that they do not contradict each other. Of course, when applicable to each other, appropriate modifications may be made to the corresponding features if necessary. The various aspects and features of the present invention are further described below.

[0044] All references cited in this invention are incorporated herein by reference in their entirety, and in the event of any inconsistency between the meanings expressed in these references and those expressed herein, the meanings expressed herein shall prevail. Furthermore, the various terms and phrases used in this invention have their general meanings known to those skilled in the art; however, this invention still seeks to provide a more detailed explanation and interpretation of these terms and phrases, and in the event of any inconsistency between the mentioned terms and their known meanings and those expressed herein, the meanings expressed herein shall prevail.

[0045] Pneumonia refers to inflammation of the terminal airways, alveoli, and pulmonary interstitium, which can be caused by pathogenic microorganisms, immune damage, physical and chemical factors, allergies, etc. In recent years, scholars at home and abroad have often used lipopolysaccharide (LPS) to induce pneumonia in animal models to simulate the pathogenesis of pneumonia. This invention uses Polygonum capitatum to investigate the efficacy of LPS-induced pneumonia in animal models for the treatment of pneumonia.

[0046] Polygonum capitatum Buch-Ham ex D. Don is a perennial herb belonging to the Polygonaceae family and the Polygonum genus. It possesses the effects of clearing heat and dampness, promoting diuresis, and relieving strangury, and has shown significant efficacy in the clinical treatment of urinary tract infections. The traditional Chinese medicine preparation "Relinqing Granules," made from Polygonum capitatum, was included in the National Basic Medical Insurance Catalog in 2004 and the Guizhou Provincial Essential Medicines Catalog in 2012.

[0047] Polygonum capitatum is a commonly used medicinal herb in ethnic minority areas, mainly used for pyelonephritis, urinary tract infections, and diuresis. Related pharmacological studies are rare, with only a few reports from Ren Guangyou et al. Ren Guangyou conducted experiments using a mouse model of bacterial pyelonephritis. The results showed that the WBC and BLD levels in the urine of mice in the Polygonum capitatum water extract group were significantly reduced compared to the control group, indicating that the Polygonum capitatum water extract has a certain anti-inflammatory effect on pyelonephritis. Ren Guangyou et al. observed the mortality of mice within 5 days after intraperitoneal injection of Escherichia coli bacterial solution. The results showed that the mortality rate in the control group was 100%, while the mortality rates in the Polygonum capitatum group were 20% and 50%, respectively, indicating that the Polygonum capitatum water extract can combat infections caused by Escherichia coli. Ren Guangyou et al. administered Polygonum capitatum water extract by gavage to rabbits. The results showed no significant difference in body temperature between the Polygonum capitatum water extract group and the control group, but it could reduce the fever induced by intravenous typhoid and paratyphoid vaccines in rabbits. Ren Guangyou et al. administered water extracts of Polygonum capitatum to rabbits and mice via gavage, comparing urine output with a blank control group and a furosemide control group. The results showed that the water extracts of Polygonum capitatum had no significant diuretic effect on rabbits and mice. Xu Yingchun et al. used the agar dilution method to detect the in vitro antibacterial activity of Polygonum capitatum against 10 strains of Neisseria gonorrhoeae (gonococci). The results showed that Polygonum capitatum had antibacterial activity against Neisseria gonorrhoeae. The minimum inhibitory concentration (MIC) against the 10 strains of Neisseria gonorrhoeae ranged from 8 to 32 g / L, with an average value of 11.2 g / L.

[0048] The method of the present invention can exhibit one or more superior effects. Attached Figure Description

[0049] Figure 1 Effects of Relinqing granules on pathological changes in lung tissue of mice with LPS-induced acute pneumonia (×400). Detailed Implementation

[0050] The present invention can be further described by the following embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. The present invention generally and / or specifically describes the materials and test methods used in the experiments. Although many materials and operation methods used to achieve the purpose of the present invention are well known in the art, the present invention still describes them in as much detail as possible herein. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0051] In the present invention, unless otherwise stated, the Herlingqing granules used are prepared according to the following method: Take 1250 g of Polygonum capitatum, decoct twice with water for 1.5 hours each time. Filter the decoction, combine the filtrates, concentrate to an appropriate amount, filter, spray dry, mix evenly with an appropriate amount of soluble starch, make into granules, dry, and make 500 g, thus obtaining the product. Of course, the present invention can also use the sugar-containing Herlingqing granules. These Herlingqing granules comply with the regulations of the same-named variety described in the first part of the Chinese Pharmacopoeia 2020 edition.

[0052] Example 1: Protective effect of Herlingqing granules on LPS-induced acute pneumonia in mice

[0053] In this experiment, a pneumonia model of BALB / c mice was induced by LPS. The mice were randomly divided into a normal group, a model group, a positive control group, and high, medium, and low dose groups of Herlingqing granules, with 12 mice in each group. The lung index of each group of mice was detected, the pathological changes of the lung tissue of the mice were detected by HE staining, and the contents of TNF-α, IL-1, IL-6, and PGE2 in the bronchoalveolar lavage fluid were measured by ELISA.

[0054] 1. Experimental materials

[0055] (1) Experimental animals

[0056] Healthy SPF-grade BALB / c mice, half male and half female, weighing 18 - 22 g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., license number: SCXK(Xiang)2019 - 0004.

[0057] (2) Main instruments

[0058] Compressed atomizer (Oberthur, CNB69011),

[0059] Centrifuge (Nest, 2015002),

[0060] Refrigerator (Haier, BCD-215KAN DZ),

[0061] Vertical ultra-low temperature storage box (Haier, DW-86L386)

[0062] Pipettes (10~100μl, Dalong, KA0005852)

[0063] Pipettes (100~1000μl, Dalong, DX64849)

[0064] Beijing Top (DEM-3, Automatic Plate Washing Machine)

[0065] BIOBASE (EL10A, automated microplate reader).

[0066] (3) Main reagents

[0067] Relinqing Granules (Guizhou Weimen Pharmaceutical Co., Ltd., 210602)

[0068] Dexamethasone Acetate Tablets (Chang Le Pharmaceutical, D2104121)

[0069] LPS (Lipopolysaccharide, SIGMA, L9143)

[0070] Mouse Tumor Necrosis Factor α (TNF-α) ELISA Kit (Genemax, Catalog No. JYM0218Mo / Lot No. GR20220610)

[0071] Mouse IL-1β ELISA Kit (Genemax, catalog number JYM0531Mo / lot number GR20220610)

[0072] Mouse IL-6 ELISA Kit (Genemax, catalog number JYM0012Mo / lot number GR20220610)

[0073] Mouse prostaglandin E2 (PGE2) ELISA kit (GeneMed, catalog number JYM0603Mo / lot number GR20220610).

[0074] (4) Reagent preparation

[0075] LPS solution preparation: Dilute 150 mg of LPS to a 15 ml volumetric flask and store at 4 °C.

[0076] Preparation of Relinqing Granule Suspension: Take an appropriate amount of Relinqing granules and grind them in a mortar. Take 41.6g of the ground powder and dilute it to 100ml volumetric flask with physiological saline. This is the high dose. Take 40ml of the high dose solution and add 40ml of physiological saline. Shake well. This is the medium dose. Take 20ml of the medium dose solution and add 20ml of physiological saline. Shake well. This is the low dose.

[0077] Preparation of positive control solution: Grind an appropriate amount of dexamethasone tablets in a mortar, accurately weigh 8.78g of the ground powder, and dilute to 100ml in a volumetric flask (0.0878g / ml).

[0078] 2. Experimental Methods

[0079] (1) Grouping, modeling and drug administration

[0080] Healthy BALB / c mice, half male and half female, were randomly divided into 6 groups: blank control group, model group, high-dose Relinqing granules group (6.24 g / kg), medium-dose Relinqing granules group (3.12 g / kg), low-dose Relinqing granules group (1.56 g / kg), and positive control group (dexamethasone 0.878 mg / kg / d), with 12 mice in each group.

[0081] Adaptive feeding (laboratory environment: 25±2℃, relative humidity 50±5%) for 7 days before modeling; LPS nebulization method (20mg / kg) was used for modeling, and modeling was carried out continuously for three days;

[0082] The day after modeling, the high, medium and low dose groups of Relinqing granules and the positive control group were administered the drug by gavage, while the blank control group and the model group were administered an equal volume of physiological saline by gavage, once a day for 7 consecutive days.

[0083] (2)Acquisition of materials

[0084] After blood was collected from the mouse eyeballs, the mouse was euthanized by dislocation of the neck. The blood was allowed to coagulate naturally at room temperature for 30 minutes, then centrifuged at 1000xg for about 15 minutes. The serum supernatant was collected and stored at 4°C for later testing (or frozen at -80°C).

[0085] Blood was collected from mice, and bronchoalveolar lavage fluid (frozen) and whole lung tissue (right lobe frozen, left lobe preserved in formalin) were collected for testing.

[0086] (3) Detection and data processing

[0087] (3.1) Effect of Relinqing Granules on Lung Index in LPS-Induced Acute Pneumonia Mice:

[0088] After euthanizing the mice by collecting blood from their eyeballs, the weight of their entire lungs was quickly and accurately measured, and the lung index was calculated using the following formula: Lung index = Lung weight of mouse (g) / Body weight of mouse (g);

[0089] (3.2) Effects of Relinqing Granules on the pathological changes of lung tissue in mice with LPS-induced acute pneumonia:

[0090] Lung tissue specimens were dehydrated, trimmed, embedded, sectioned, stained, mounted, and examined under a microscope according to standard pathological examination procedures.

[0091] [For example, the operating conditions for dehydrating fixed lung tissue using a fully automatic dehydrator are as follows: dehydration time: 75% alcohol 4h, 85% alcohol 2h, 95% alcohol 1h, 100% alcohol 0.5h, 100% alcohol 0.5h, 100% alcohol 0.5h, xylene 10min, xylene 10min, paraffin 1h, paraffin 2h, paraffin 3h;]

[0092] [For example, after embedding and sectioning, the following steps are performed for staining and mounting: dewaxing the sections to water, staining with hematoxylin for 10-20 min, rinsing with tap water for 1-3 min, differentiating with hydrochloric acid alcohol for 5-10 s, rinsing with tap water for 1-3 min, placing them in warm water at 50℃ or a weakly alkaline aqueous solution to turn blue until blue appears, rinsing with tap water for 1-3 min, placing them in 85% alcohol for 3-5 min, staining with eosin for 3-5 min, rinsing with water for 3-5 s, dehydrating with a gradient of alcohols, clearing with xylene, and mounting with neutral resin;]

[0093] [For example, during image acquisition, an upright fluorescence microscope (DM500) manufactured by Leica GmbH, Germany, was used to acquire images of the slides. Each slide was first observed at 200x magnification to observe the entire tissue and the gross lesions. Then, the area to be observed was selected to acquire images at 400x magnification to observe the specific lesions and to score lung injury: based on the degree of lesions such as interstitial pulmonary edema, inflammatory cell infiltration, alveolar edema, alveolar hemorrhage, atelectasis, and hyaline membrane formation, the severity was scored from 0 to 3 points, respectively, according to normal, mild, moderate, and severe.]

[0094] (3.3) Determination of the effects of Relinqing granules on the levels of IL-6, IL-1β, PGE2, and TNF-α in bronchoalveolar lavage fluid of mice with LPS-induced acute pneumonia:

[0095] The levels of TNF-α, IL-1β, LI-6, and PGE2 in the serum of mice in each group were detected using an ELISA kit: Serum from each group of mice was collected, and the levels of IL-1β, LI-6, and PGE2 in the serum of each group were detected according to the ELISA kit instructions; in addition, the TNF-α index in the serum was detected according to the method in Example 2.

[0096] All experimental data are expressed as mean ± standard deviation (s). Statistical analysis was performed using SPSS 26.0 software. One-way ANOVA was used to analyze data when comparisons among multiple groups satisfied normality and homogeneity of variance. P A value <0.05 is considered statistically significant.

[0097] Statistical results on the therapeutic effect of Relinqing granules on (acute) pneumonia mice were obtained based on the above test data.

[0098] 3. Experimental Results

[0099] (1) Effects of Relinqing Granules on Body Weight and Lung Index in LPS-Induced Acute Pneumonia Mice

[0100] The results showed no significant difference in body weight among the groups of mice after modeling. P >0.05). Compared with the blank group, the lung index of mice in the model group was significantly increased ( P <0.01); Compared with the model group, the lung index of mice in the high-dose group of Relinqing granules and the positive control group was significantly decreased ( P <0.05). See Table 1.

[0101] Table 1: Effects of Relinqing Granules on Body Weight and Lung Index in LPS-Induced Acute Pneumonia Mice (±s, n=6)

[0102]

[0103] Note: Compared with the blank group: ** P <0.01, * P <0.05; compared with the model group: ## P <0.01, # P <0.05;

[0104] (2) Effects of Relinqing Granules on Lung Tissue Pathology in Mice with LPS-Induced Acute Pneumonia

[0105] HE staining showed that in the blank control group, the visceral pleura of the lung tissue of mice was covered by a thin serous membrane, with a single layer of mesothelial cells on the outermost layer and a small amount of fibrous tissue under the mesothelial cells. The tissue structure was normal, with no obvious thickening. The bronchial branching structure under the pleura was intact, and a small number of inflammatory cells were occasionally seen in the alveoli. In the model group, a small number of inflammatory cells were seen in the bronchi, and a large number of inflammatory cells were seen in the alveoli, with a small amount of pulmonary hemorrhage. Compared with the model group, the above-mentioned pathological changes in the lung tissue of mice in each dose group of Relinqing granules and the positive control group were significantly reduced, with the highest dose group of Relinqing granules showing the mildest results. Figure 1 Furthermore, the pathological results of lung tissue in groups ⑦ and ⑧ were found to be similar to those in group ④.

[0106] (3) Effects of Relinqing Granules on the levels of IL-6, IL-1, PGE2, and TNF-α in bronchoalveolar lavage fluid of mice with LPS-induced acute pneumonia

[0107] ELISA results showed that, compared with the control group, the levels of IL-6, IL-1, PGE2, and TNF-α in the bronchoalveolar lavage fluid of mice in the model group were significantly increased. P<0.01); Compared with the model group, the levels of IL-6, IL-1, PGE2, and TNF-α in the bronchoalveolar lavage fluid of the high-dose Relinqing granules group and the positive control group were significantly decreased ( P <0.01), and the levels of IL-6, IL-1, and PGE2 in bronchoalveolar lavage fluid of the low- and medium-dose groups of Relinqing granules also decreased to varying degrees. P <0.05). See Table 2.

[0108] Table 2: Effects of Relinqing Granules on the levels of IL-6, IL-1, PGE2, and TNF-α in bronchoalveolar lavage fluid of mice with LPS-induced acute pneumonia (±s, n=6)

[0109]

[0110] Note: Compared with the blank group: ** P <0.01, * P <0.05; compared with the model group: ## P <0.01, # P <0.05;

[0111] Example 2: Measurement of TNF-α concentration in mouse serum

[0112] In Example 2, a TNF-α ELISA kit was used to detect the concentration of TNF-α in mouse serum. The TNF-α ELISA kit used in this study was the commercially available JYM0218Mo mouse tumor necrosis factor α (TNF-α) ELISA kit. This TNF-α ELISA kit can be used for the in vitro quantitative detection of tumor necrosis factor α (TNF-α) in mouse serum, plasma, tissue, cell supernatant, and related liquid samples. The specificity of the TNF-α ELISA kit is that it can detect mouse tumor necrosis factor α (TNF-α) in samples and has no significant cross-reactivity with other related proteins. The repeatability of the TNF-α ELISA kit is as follows: intra-plate coefficient of variation ≤9% (generally, intra-plate coefficient of variation ≤3% is preferred), inter-plate coefficient of variation ≤13% (generally, inter-plate coefficient of variation ≤6% is preferred). The detection range of this kit is 8 pg / ml-500 pg / ml, and the method sensitivity is ≤1.2 pg / ml.

[0113] The experimental principle of the TNF-α ELISA kit is to determine the level of mouse tumor necrosis factor α (TNF-α) in samples using a double-antibody sandwich method. Purified mouse tumor necrosis factor α (TNF-α) antibody is coated onto microplates to prepare a solid-phase antibody. Tumor necrosis factor α (TNF-α) is added sequentially to the microplates coated with monoclonal antibody, followed by binding with HRP-labeled tumor necrosis factor α (TNF-α) antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted to blue under the catalysis of HRP enzyme, and then to yellow under acidic conditions. The color intensity is positively correlated with the tumor necrosis factor α (TNF-α) level in the sample. The absorbance (OD value) is measured at 450 nm using an ELISA reader, and the concentration of mouse tumor necrosis factor α (TNF-α) in the sample is calculated using a standard curve.

[0114] The TNF-α ELISA kit consists of the following components: 1 instruction manual, 2 sealing films (96), 1 ELISA plate (96 wells, 12 wells × 8 strips), 1 bottle of 6ml enzyme-labeled reagent, 1 bottle of 0.5ml standard (720pg / ml), 1 bottle of 1.5ml standard diluent, 1 bottle of 6ml sample diluent, 1 bottle of 6ml Substrate Solution A, 1 bottle of 6ml Substrate Solution B, 1 bottle of 6ml Stop Solution, 1 bottle of 20ml Wash Buffer (30x), and 2 sealing films (96).

[0115] There are general requirements for sample preparation when using the TNF-α ELISA kit described in this article. For serum: Collect blood using a blood collection tube, allow the blood to clot naturally at room temperature for 30 minutes, centrifuge at 1000xg for about 15 minutes, collect the serum supernatant, and store at 4°C until testing, or aliquot and store at -20°C or -80°C.

[0116] In Example 2, the concentration of TNF-α in mouse serum was detected using a TNF-α ELISA kit. The procedure is as follows:

[0117] a. Dilution of standard products:

[0118] Take six small test tubes and number them sequentially. First, add 100µl of standard diluent to each test tube. Then, add 100µl of the original concentration standard to the first test tube and mix thoroughly. Next, add 100µl of the original concentration standard to the second test tube and mix thoroughly. Then, add 100µl of the original concentration standard to the third test tube and mix thoroughly. Next, add 100µl of the original concentration standard to the fourth test tube and mix thoroughly. Finally, add 100µl of the original concentration standard to the fifth test tube and mix thoroughly. Add µl to the fifth test tube and mix thoroughly; then take 100µl from this test tube and discard it; the sixth test tube is used as standard 0, and the concentrations of each diluted tube are 360pg / ml, 180pg / ml, 90pg / ml, 45pg / ml, 22.5pg / ml, and 0pg / ml, respectively; set up standard wells on the enzyme-labeled plate, and add 50µl of different concentrations of standard in sequence, with 5 parallel wells for each concentration;

[0119] b. Sample preparation and loading: Collect blood using blood collection tubes, allow the blood to coagulate naturally at room temperature for 30 minutes, centrifuge at 1000xg for approximately 15 minutes, collect the supernatant (serum), and mix the supernatant with the additional solution at a volume ratio of 9:1 to obtain the serum sample to be tested; set up sample wells and blank control wells (no sample or enzyme-labeled reagent is added to the blank control wells, and the remaining steps are the same as for the sample wells); add 40 μl of sample diluent to the sample wells on the enzyme-labeled plate, and then add 10 μl of the sample to be tested; when loading the sample, add the sample to the bottom of the well, avoiding contact with the well wall as much as possible, and gently shake to mix; the additional solution is an aqueous solution containing 1.25% glycine and 0.2% zinc chloride;

[0120] c. Incubation: Add 50 μl of enzyme-labeled reagent to each well, except for the blank control wells; seal the plate with sealing film and incubate at 37°C for 30 minutes;

[0121] d. Washing: Dilute the 30x concentrated washing solution with distilled water to obtain the washing solution; carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry;

[0122] d. Color development: Add 50 μl of color reagent A to each well, then add 50 μl of color reagent B, gently shake to mix, and develop color at 37°C in the dark for 10 minutes; then add 50 μl of stop solution to each well to stop the reaction, at which point the blue color will turn yellow.

[0123] e. Measurement and Calculation: Within 15 minutes of adding the stop solution, zero the instrument using the blank control wells and sequentially measure the absorbance (OD) value of each well at 450 nm using a microplate reader. Calculate the linear regression equation of the standard curve using the concentration and OD value of the standards, and calculate the sample concentration using the sample OD value. Note that the dilution factor with the additional solution should be considered when calculating the TNF-α concentration in serum samples.

[0124] The results of determining the serum TNF-α concentration in mice with LPS-induced acute pneumonia in Example 1 using the method of Example 2 are shown in Table 2 above (the results for each group are ±s results for 12 animals). Additionally, when determining TNF-α using the method of Example 2 in Table 2 above, the serum of 12 animals in each group was mixed in equal proportions to obtain a mixed serum (which can be referred to as mixed serum X in this document). This mixed serum was used to measure TNF-α using the TNF-α ELISA kit of Example 2 to examine the repeatability characterized by intra-plate coefficient of variation (intra-plate CV, statistical analysis of 5 parallel tests within the same plate, the same below) and inter-plate coefficient of variation (inter-plate CV, statistical analysis of 5 parallel tests between the same plates, the same below). The results showed that the intra-plate CVs for groups ① to ⑥ were 3.17%, 3.04%, 3.76%, 2.94%, 3.85%, and 3.47%, respectively, and the inter-plate CVs for groups ① to ⑥ were 5.63%, 6.13%, 6.06%, 5.36%, 5.94%, and 6.32%, respectively; all groups showed excellent repeatability of the measurement results. However, it has been found that when the above-mentioned additional solution is not premixed with the serum, the TNF-α ELISA test shows significantly worse CV results for serum from animals treated with *Heat-Lymphoid Escherichia coli*, which does not meet the requirements of general ELISA assays. This result is presented in Example 3 below. It should be noted that this phenomenon of poor CV results for TNF-α, which is only present in the serum of *Heat-Lymphoid Escherichia coli*, was not observed when measuring other indicators using other ELISA kits in this invention.

[0125] Example 3: Referring to Examples 1 and 2, the same TNF-α ELISA kit was used to detect the intra- and inter-plate coefficients of variation (CVs) of mouse mixed serum X in the same method. The only difference was that the additional solution used in step "b. Preparation and addition of test samples" in Example 2 was an aqueous solution containing 1.25% glycine. The results showed that the intra-plate CVs for the six groups ① to ⑥ were 3.86%, 3.47%, 3.11%, 16.53%, 14.35%, and 14.63%, respectively, and the inter-plate CVs for the six groups ① to ⑥ were 5.66%, 6.13%, 5.85%, 17.34%, 15.31%, and 14.13%, respectively. This indicates that the repeatability of CV characterization in this ELISA method for mouse serum administered with hot lysin was not ideal.

[0126] Example 4: Referring to Examples 1 and 2, the same TNF-α ELISA kit was used to detect the intra- and inter-plate coefficients of variation (CVs) of mouse mixed serum X in the same method. The only difference was that the additional solution used in step "b. Preparation and addition of test samples" in Example 2 was an aqueous solution containing 0.2% zinc chloride. The results showed that the intra-plate CVs for the six groups ① to ⑥ were 4.03%, 3.87%, 3.72%, 16.12%, 15.53%, and 13.14%, respectively, and the inter-plate CVs for the six groups ① to ⑥ were 5.32%, 4.82%, 5.11%, 16.78%, 14.63%, and 12.11%, respectively. This indicates that the repeatability of CV characterization in this ELISA method for mouse serum administered with heat-induced leukemia was not ideal.

[0127] Example 5: Referring to Examples 1 and 2, the same TNF-α ELISA kit was used to detect the intra- and inter-plate coefficients of variation (CVs) of mouse mixed serum X in the same method. The only difference was that in step "b. Sample preparation and addition", the mixed serum X was measured directly without being mixed with the additional solution. The results showed that the intra-plate CVs for the six groups ① to ⑥ were 4.63%, 4.13%, 3.93%, 15.89%, 15.23%, and 13.74%, respectively, and the inter-plate CVs for the six groups ① to ⑥ were 5.65%, 5.13%, 5.02%, 17.54%, 16.13%, and 13.27%, respectively. This indicates that the repeatability of CV characterization in this ELISA method for mouse serum administered with heat-induced leukemia was not ideal.

[0128] Example 6: The effect of Relinqing granules in treating pneumonia

[0129] Example 1 of this invention investigated and demonstrated the excellent therapeutic effect of Relinqing granules on related diseases, and Example 6 extends this therapeutic effect through further research.

[0130] Example 6 is an additional test group that was performed in parallel with the experiment in Example 1 (these test groups were not described in Example 1, but are supplemented in Example 6), namely, the addition of two test groups of hot rinsing granules numbered ⑦ and ⑧ as follows:

[0131] ⑦ Medium-dose group of Relinqing granules: Relinqing granules 3.12g / kg + ferrous gluconate 60mg / kg,

[0132] ⑧ Low-dose group of Relinqing granules: 1.56g / kg of Relinqing granules + 30mg / kg of ferrous gluconate.

[0133] The ferrous gluconate tablets used in the experiment were tablets (Kangnuo Pharmaceutical) with the national drug approval number H41023259. During the experiment, the drugs were within their expiration date. Ferrous gluconate tablets and Relinqing granules were mixed together in a certain proportion to prepare the drug solution for administration.

[0134] The results for groups ⑦ and ⑧ are as described in Example 1. Furthermore, in Example 2, when TNF-α was measured, the intra-plate CVs for groups ⑦ and ⑧ were 3.44% and 3.09%, respectively, and the inter-plate CVs were 6.42% and 5.84%, respectively, demonstrating excellent methodological characteristics.

[0135] The results of this embodiment indicate that when Relinqing granules / ferrous gluconate are administered in a combination ratio of 3.12g / 60mg, ferrous gluconate can improve the therapeutic effect of Relinqing granules, especially the therapeutic effect characterized by TNF-α serum concentration.

[0136] Therefore, in any embodiment of any aspect of the present invention, ferrous gluconate is applied simultaneously with the administration of Relinqing granules to the organism, and the weight ratio of Relinqing granules to ferrous gluconate is 48 to 52:1, for example, 52:1; for example, in terms of the pharmaceutical use of the present invention, the medicine for treating the corresponding disease further contains the above-mentioned proportion of ferrous gluconate.

[0137] Polygonum capitatum is the main ingredient of Relinqing granules. It is the whole herb of Polygonum capitatum, a plant of the Polygonaceae family. According to literature, it tastes bitter and pungent, and is warm and neutral in nature. It clears heat and detoxifies to treat urinary tract infections [Edited by the Health Management Service Station of the Revolutionary Committee of Guangxi Zhuang Autonomous Region. Guangxi Traditional Chinese Medicine, Volume 2 [M]. Nanning: Guangxi People's Publishing House. 1970]. At present, the research on Relinqing granules mainly focuses on its effect on urinary tract infections. A large number of animal experiments and clinical studies have proved that Relinqing granules have significant efficacy and have become one of the first-line drugs for the clinical treatment of urinary tract infections. Modern research has found that it also has a certain therapeutic effect on various inflammations such as gastritis and nephritis, and inflammation often causes fever. Traditional Chinese medicine believes that fever is the result of the struggle between pathogenic factors and the body's vital energy after the invasion of external pathogens. It often uses drugs that clear heat in the upper burner to relieve fever. Traditional Chinese medicine for treating urinary tract infections in the lower burner often has the effect of "clearing heat from the upper to the lower" [Kang Kaibiao, Liu Shuying, Li Shuling, et al. Professor Wang Zili's experience in treating urinary tract infections by clearing heat from the upper to the lower [J]. [Western Journal of Traditional Chinese Medicine, 2021, 34(02): 62-63], therefore it is speculated that Relinqing Granules have the effect of clearing heat in the upper burner. In 2020, Relinqing Granules were included in the "Guizhou Province COVID-19 Traditional Chinese Medicine Rehabilitation Program" by the Guizhou Provincial Administration of Traditional Chinese Medicine. Therefore, our research group intends to observe the antipyretic effect of Relinqing Granules and explore its mechanism of action to provide more options for clinical practice.

[0138] Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, consisting of three parts: lipid A, core oligosaccharide, and O-specific chain. The core oligosaccharide connects the hydrophilic O-specific chain in the outer layer to the hydrophobic lipid A in the inner layer. The core oligosaccharide is a branched oligosaccharide chain composed of 9-10 glycosyl groups, which can be further divided into inner and outer core oligosaccharides. The inner core oligosaccharide attaches to lipid A via acid-labile ketoglycosidic bonds. The outer core oligosaccharide is mainly composed of neutral and basic hexoses and is linked to the O-specific chain. Differences in monosaccharide composition and conformation exist among different bacterial strains, which are fundamental to determining the LPS core type. When bacteria invade the human body, they release their surface LPS. LPS first binds to lipopolysaccharide-binding protein (LBP), which transports LPS to the membrane surface of immune cells, where it binds to the membrane protein CD14. LPS is not released by bacteria under normal living conditions, but it is released after bacterial cell death and rupture, artificial lysis, or active cell growth and reproduction. While it is not toxic in itself, as a non-specific immunogen, it interacts with host effector cells (mainly monocytes, macrophages, and neutrophils) after entering the microcirculation, secreting bioactive molecules such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), reactive oxygen species (ROS), and NO. This can cause clinical syndromes such as fever, disseminated intravascular coagulation, multiple organ failure, and shock. LPS toxicity occurs through the formation of a protective barrier around bacteria to evade antibiotics, acting on host cells to produce inflammatory cytokines, disrupting the internal environment and causing diseases such as endotoxemia and sepsis. The activity of LPS is mainly due to its immune-activating effect, which can have a variety of biological functions such as anti-tumor, anti-radiation, anti-infection, promoting the occurrence of secondary cataracts, promoting periodontal inflammation, promoting pericardial cell proliferation, and relieving asthma.

[0139] In summary, this invention experimentally explores the protective effect and mechanism of Relinqing granules against LPS-induced acute pneumonia in mice. Specifically, the method involves using an LPS-induced BALB / c mouse pneumonia model, randomly dividing mice into a blank group, a model group, a positive control group, and high, medium, and low dose Relinqing granule groups, with 12 mice in each group. Lung index was measured in each group, HE staining was used to detect pathological changes in mouse lung tissue, and ELISA was used to determine the levels of TNF-α, IL-1, IL-6, and PGE2 in bronchoalveolar lavage fluid. In this experiment, the lung index and the levels of IL-6, IL-1, PGE2, and TNF-α in the bronchoalveolar lavage fluid of the model group mice were significantly higher than those in the control group. After treatment with high doses of Relinqing granules, the lung index and the levels of IL-6, IL-1, PGE2, and TNF-α in the bronchoalveolar lavage fluid of the mice were significantly lower than those in the model group. After treatment with medium and low doses of Relinqing granules, the levels of IL-6, IL-1, and PGE2 in the bronchoalveolar lavage fluid of the mice also decreased to varying degrees. This suggests that Relinqing granules can inhibit the production of inflammatory factors, thereby playing a role in the treatment of pneumonia, and that the therapeutic effect has a dose-response relationship within a certain range.

[0140] The results of this study show that Relinqing granules can alleviate the symptoms of pneumonia, such as acute pneumonia, and improve related serum indicators, indicating that Relinqing granules are effective in treating pneumonia.

[0141] The embodiments described herein are merely illustrative of the composition and effects of the present invention and are not intended to limit the patent scope of the present invention. Therefore, those skilled in the art can make similar modifications without departing from the structure of the present invention, and all such modifications fall within the patent scope of the present invention. These modifications should also be considered within the protection scope of the present invention and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. The use of Relinqing granules in the preparation of drugs for treating pneumonia; said Relinqing granules are granules made from Polygonum capitatum; said granules are prepared by the following method: take 1250g of Polygonum capitatum, add water and decoct twice, 1.5 hours each time, filter the decoction, combine the filtrates, concentrate to an appropriate amount, filter, spray dry, mix with an appropriate amount of soluble starch, make into granules, dry, and make 500g, which is the result.

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