Preparation method of medicine for treating sepsis lung injury

Abstract

A medicine for treating sepsis lung injury relates to the technical field of medical treatment, the medicine comprises guaijave glycoside, dimethyl sulfoxide and sterile corn oil, the medicine comprises 10 mg of guaijave glycoside, 100 [mu] l of dimethyl sulfoxide and 900 [mu] l of sterile corn oil, and the medicine is used for treating sepsis lung injury. Through carefully selected medicine components and proportions, inflammatory factors of mice suffering from sepsis lung injury can be effectively inhibited, injury of lung tissues is reduced, and then the survival rate of the mice suffering from sepsis is increased.

Description

Technical field:

[0001] The invention relates to the field of medical technology, and in particular to a method for preparing a medicine for treating sepsis-induced lung injury. Background technology:

[0002] Sepsis refers to life-threatening organ dysfunction caused by host response disorders caused by infection. There are up to 11 million sepsis-related deaths worldwide each year. Pathogen infection is an important cause of sepsis. At present, due to its high morbidity and mortality, it has become an important public health problem facing the world. There is an inflammatory factor storm (i.e., excessive inflammatory response) and immune suppression in the disease process of sepsis patients. Excessive inflammatory response is an important cause of lung damage. Improving lung tissue damage can effectively improve the survival rate of sepsis patients. Early antibiotic anti-infection is an important means of treating septic lung injury. At present, bacterial resistance and the slow development of new antibiotics have led to the dilemma of no drugs available for clinical treatment of septic lung injury. In order to solve the above problems, a new technical solution is proposed. Summary of the invention:

[0003] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and to propose a preparation method and application of a drug for treating sepsis-induced lung injury.

[0004] The technical solution adopted by the present invention is: a drug for treating septic lung injury, the drug comprising guava glycosides, dimethyl sulfoxide, and sterile corn oil, and the proportions of the components of the drug are 10 mg of guava glycosides, 100 ul of dimethyl sulfoxide, and 900 ul of sterile corn oil.

[0005] As a preferred technical solution, the guava glycoside is quercetin-3-O-α-L-arabinoside, which is extracted from the traditional Chinese medicine monomer of Oroxylum indica. The drug number is A1157, which is yellow powder, easily soluble in dimethyl sulfoxide, slightly soluble in formaldehyde, and has a drug purity of more than 98%. The monomer molecular formula is C 20 H 18 O 11 , the molecular weight is 434.35. As a preferred technical solution, the control drug solution is prepared by mixing dimethyl sulfoxide and corn oil in a ratio of 1 to 9.

[0006] A method for preparing a drug for treating sepsis-induced lung injury, the method comprising the following steps:

[0007] Step 1, preparing control drugs;

[0008] Step 2, preparing guava glycoside drug solution;

[0009] Step 3: Use cecal ligation and puncture (CLP) to establish a septic lung injury model.

[0010] As a preferred technical solution, the control drug is prepared by mixing dimethyl sulfoxide and sterile corn oil in a ratio of 1 to 9 to obtain a drug solvent (solvents), i.e., the control drug;

[0011] As a preferred technical solution, the preparation of guava glycosides comprises first dissolving 10 mg of guava glycosides in 100 ul of dimethyl sulfoxide, then adding 900 ul of sterile corn oil, vortexing and mixing with a stirrer, and finally preparing a drug with a guava glycoside concentration of 10 mg / ml.

[0012] A drug for treating sepsis lung injury is used to establish a sepsis lung injury model. A mouse of 6-8 weeks old and weighing 20-22g is taken, and 200ul of 5% chloral hydrate is injected into the mouse's abdominal cavity. After anesthesia takes effect, the mouse's abdomen is disinfected with 75% ethanol, the skin is lifted with sterile ophthalmic forceps, and the skin and peritoneum are cut in sequence on the left side of the mouse's mid-abdomen with sterile ophthalmic scissors. The wound is about 0.5cm long, the mouse's cecum is pulled out with ophthalmic forceps, and feces in the cecum are squeezed into the distal end with forceps, and the cecum is ligated with No. 4 surgical suture at 2 / 3 of the cecum, and the cecum is punctured with an 18-gauge syringe needle and passed out from the opposite side, a small amount of feces is squeezed out, the cecum is sent into the abdominal cavity, the abdomen is sutured, and the skin and peritoneum are cut in sequence on the left side of the mouse's mid-abdomen with sterile ophthalmic scissors. The wound is about 0.5cm long, the mouse's cecum is pulled out with ophthalmic forceps, and then the cecum is sent into the abdominal cavity, the abdomen is sutured, and then 1ml is subcutaneously injected into the back of the mouse. Fluid resuscitation was performed with 0.9% NaCl, and death was observed within 2 hours after complete resuscitation. Surviving mice were divided into groups.

[0013] The beneficial effects of the present invention are: the drug for treating septic lung injury can effectively inhibit the inflammatory factor storm of sepsis patients, reduce lung tissue damage, and thus improve the survival rate of septic mice through carefully selected drug ingredients and ratios. Among them, guava glycosides, as the main component of the drug, not only have good anti-inflammatory effects, but also can effectively improve immune function and inhibit excessive inflammatory reactions. At the same time, dimethyl sulfoxide and sterile corn oil are used as solvents to provide a stable carrier environment for the drug, which can enhance the solubility and permeability of the drug, so that the drug can enter the body faster and play a role. This method can not only ensure the stability and purity of the drug, but also improve the preparation efficiency and reduce costs. At the same time, the present invention also provides an application method of the drug on a mouse model, which provides strong support for further research and application of the drug.

[0014] In terms of preparation, the present invention adopts a simple and easy preparation method, using dimethyl sulfoxide and sterile corn oil as drug solvents. This method can not only ensure the stability and purity of the drug, but also improve the preparation efficiency and reduce the cost. At the same time, the present invention also provides an application method of the drug on a mouse model, which provides strong support for further research and application of the drug. Description of the drawings:

[0015] Figure 1 This is a graph showing that guajaroside improves the survival of septic mice;

[0016] Figure 2 This is a diagram showing that guajaroside of the present invention reduces lung tissue damage;

[0017] Figure 3 This is a diagram showing that guajava glycosides of the present invention inhibit inflammatory response in lung tissue. Specific implementation method:

[0018] The present invention is further described with reference to specific embodiments:

[0019] A drug for treating septic lung injury, the drug includes guava glycoside, dimethyl sulfoxide, and sterile corn oil, and the proportions of the components of the drug are 10 mg of guava glycoside, 100 ul of dimethyl sulfoxide, and 900 ul of sterile corn oil. As a preferred technical solution, the guava glycoside is quercetin-3-O-α-L-arabinoside, a Chinese medicine monomer extracted from Oroxylum indica, purchased from Chengdu Muenster Biotechnology Co., Ltd., the drug number is A1157, it is a yellow powder, slightly soluble in formaldehyde, easily soluble in dimethyl sulfoxide, the drug purity is greater than 98%, the monomer molecular formula is C20H18O11, and the molecular weight is 434.35. As a preferred technical solution, the sterile corn oil is prepared by mixing dimethyl sulfoxide and corn oil in a ratio of 1 to 9.

[0020] A method for preparing a drug for treating sepsis-induced lung injury, the method comprising the following steps:

[0021] Step 1, preparing a control drug; as a preferred technical solution, the control drug is prepared by mixing dimethyl sulfoxide and corn oil in a ratio of 1 to 9 to obtain a control drug;

[0022] Step 2, preparing guava glycosides; as a preferred technical solution, the preparation of guava glycosides is to first dissolve 10 mg of guava glycosides in 100 ul of dimethyl sulfoxide, then add 900 ul of sterile corn oil, vortex stir and mix by a stirrer, and finally prepare a drug solution with a concentration of 10 mg / ml.

[0023] An application of a drug for treating sepsis lung injury, after preparing the drug, a CLP sepsis lung injury model needs to be established, and the model includes the following four steps:

[0024] Step 1: Anesthetize mice aged 6-8 weeks and weighing 20-22 g, and inject 200 ul of 5% chloral hydrate into the mouse's peritoneum;

[0025] Step 2: CLP group, after anesthesia takes effect, use 75% ethanol to disinfect the abdomen of the mouse. Use sterile ophthalmic forceps to lift the skin, and use sterile ophthalmic scissors to cut the skin and peritoneum in turn on the left side of the mouse's mid-abdomen. The wound is about 0.5 cm long. Use ophthalmic forceps to pull out the mouse's cecum, and use forceps to squeeze the feces in the cecum into the distal end. Use No. 4 surgical suture to ligate the cecum at 2 / 3 of the cecum (away from the ileocolic junction). Use an 18-gauge syringe needle to puncture the cecum and pass it out from the opposite side, squeeze out a little feces, send the cecum into the abdominal cavity, and suture the abdomen. Sham group (sham): Use sterile ophthalmic scissors to cut the skin and peritoneum in turn on the left side of the mouse's mid-abdomen. The wound is about 0.5 cm long. Use ophthalmic forceps to pull out the mouse's cecum, and then send the cecum into the abdominal cavity and suture the abdomen;

[0026] Step 3: Resuscitation: 1 ml of 0.9% NaCI was injected subcutaneously on the back of each mouse for fluid resuscitation;

[0027] Step 4: After the mice are fully resuscitated, observe whether there is any death within 2 hours, and the surviving mice are used for group experiments.

[0028] After the CLP sepsis model is established, the next step is grouping, which includes the following two steps:

[0029] Step 1. Survival experiment grouping: CLP mice were divided into CLP+solvents group, CLP+Gua (50 mg / kg) group, CLP+Gua (25 mg / kg) group, and CLP+Gua (12.5 mg / kg) group, with 5 mice in each group. Solvents is the control drug, i.e., the drug solvent; CLP is the cecal ligation and puncture method.

[0030] Step 2: Evaluation of the degree of lung tissue injury Grouping: The mice were divided into sham+solvents group, CLP+solvents group, sham+Gua (50 mg / kg), and CLP+Gua (50 mg / kg) group. There were 4 mice in each group. Among them, sham was a sham operation group, and CLP was a cecal ligation and puncture method.

[0031] After grouping, medication is carried out, and the medication is divided into the following steps:

[0032] Step 1: The mice in the CLP+Gua (50 mg / kg) group, the CLP+Gua (25 mg / kg) group, and the CLP+Gua (12.5 mg / kg) group were subcutaneously injected with 200 ul of guajava glycoside solution with drug concentrations of 10 mg / ml, 5 mg / ml, and 2.5 mg / ml on the back;

[0033] Step 2: 200ul of control solvents were injected subcutaneously on the back of mice in the CLP+solvents group;

[0034] Step 3: 200ul of drug with a concentration of 10mg / ml was injected subcutaneously on the back of the sham+Gua (50mg / kg) and CLP+Gua (50mg / kg) groups;

[0035] Step 4: 200ul of control drug solvents were subcutaneously injected into the back of mice in the sham+solvents group and the CLP+solvents group;

[0036] After the above experiments were completed, the survival status of the mice was observed. After the drugs were injected into the back of the mice, the survival of the mice in each group was observed and recorded every 1 hour;

[0037] HE staining was used to evaluate the degree of lung injury. The right lower lobe of the mice was removed after anesthesia and fixed with 4% paraformaldehyde for 24 h. The samples were dehydrated, embedded in paraffin, and sliced ​​(5 μm thick) for HE staining. The morphological changes, tissue structure, interstitial thickening, and perivascular inflammatory cell infiltration of each group were observed under a light microscope, and semi-quantitative analysis was performed using a scoring method.

[0038] qPCR was used to detect the mRNA transcription level. After the mouse was anesthetized, the left lung was removed, rinsed thoroughly in pre-cooled PBS, and the tissue was ground on ice to make a homogenate. The tissue homogenate was transferred into a 1.5ml EP tube and centrifuged (300g, 5min). The supernatant was discarded, 1ml trizol was added, and the cells were repeatedly blown and lysed with a pipette. The samples were frozen in a -80℃ refrigerator for later use. The mRNA transcription level of AhR was detected according to the fluorescent quantitative PCR method established in the laboratory. The main steps include RNA extraction, cDNA synthesis, real-time quantitative PCR, and analysis of the calculation results of the target gene amount of each sample with internal reference correction; the primer sequences used are shown in the following table:

[0039]

[0040] Finally, statistical analysis was performed. The experimental data and results of the present invention were analyzed using Prism8.0 statistical software, and the data were expressed as mean ± standard mean error. One-way analysis of variance was used. P < 0.05 indicates that the difference is statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001; NS P > 0.05, there is no statistical difference, and the following results were obtained. In the CLP-induced sepsis model, guava glycosides have the effect of protecting the survival of mice, and have a dose effect; in the CLP-induced sepsis model, guava glycosides have the effect of inhibiting lung inflammation and neutrophil infiltration, and protecting mice from lung tissue damage;

[0041] Reference Figure 1 In the CLP-induced sepsis mouse model, 12.5 mg / kg and 25 mg / kg guajava glycosides could not improve the survival of mice, but 50 mg / kg guajava glycosides could improve the survival of mice.

[0042] Reference Figure 2 In the CLP-induced sepsis mouse model, the CLP+solvents group significantly increased the permeability of alveolar capillary membranes and neutrophil infiltration compared with the sham+solvent group. The CLP+Gua (50 mg / kg) group showed a decrease in alveolar permeability and neutrophil infiltration.

[0043] Reference Figure 3 In the CLP-induced sepsis mouse model, compared with the sham+solvent group, the CLP+solvents group could promote the mRNA expression of inflammatory factors IL-6, TNF-α, IL-27 and chemokine CXCR4, while CLP+Gua could inhibit the mRNA expression of inflammatory factors IL-6, TNF-α, IL-27 and chemokine CXCR4. This indicates that guava glycosides can inhibit the inflammatory response of sepsis lung tissue and reduce neutrophil chemotaxis.

[0044] Through the above experiments, a drug for treating septic lung injury can be obtained. Through carefully selected drug ingredients and ratios, the inflammatory factor storm of sepsis patients can be effectively inhibited, the damage to lung tissue can be reduced, and the survival rate of sepsis patients can be improved. Among them, guava glycosides, as the main component of the drug, not only have good anti-inflammatory effects, but also can effectively improve immune function and inhibit excessive inflammatory reactions. At the same time, dimethyl sulfoxide and sterile corn oil are used as solvents to provide a stable carrier environment for the drug, which can enhance the solubility and permeability of the drug, so that the drug can enter the body faster and play a role. This method can not only ensure the stability and purity of the drug, but also improve the preparation efficiency and reduce costs. At the same time, the present invention also provides an application method of the drug on a mouse model, which provides strong support for further research and application of the drug.

[0045] For those skilled in the art, various other corresponding changes and modifications can be made according to the above technical solutions and concepts, and all of these changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A drug for treating sepsis-induced lung injury, characterized in that: The drug comprises guava glycoside (Gua), dimethyl sulfoxide (DMSO) and sterile corn oil, wherein each component of the drug is 10 mg of guava glycoside, 100 ul of dimethyl sulfoxide and 900 ul of sterile corn oil, wherein the dimethyl sulfoxide and sterile corn oil are used as drug solvents and control drugs.

2. The drug for treating sepsis-induced lung injury according to claim 1, characterized in that: The guava glycoside is quercetin-3-O-α-L-arabinoside, a Chinese medicine monomer extracted from Oroxylum indica, with a drug number of A1157, a yellow powder, slightly soluble in formaldehyde, and easily soluble in dimethyl sulfoxide. The drug purity is greater than 98%, and the monomer molecular formula is C 20 H 18 O 11 , molecular weight is 434.

35.

3. The drug for treating septic lung injury according to claim 1, wherein the drug solvent is prepared by mixing dimethyl sulfoxide and sterile corn oil in a ratio of 1 to 9.

4. The drug for treating sepsis-induced lung injury according to claim 1, characterized in that: The drug solution is prepared by dissolving 10 mg of Gua in 100 ul of dimethyl sulfoxide and then adding 900 ul of sterile corn oil.

5. A method for preparing a drug for treating sepsis-induced lung injury, characterized in that: The method comprises the following steps: Step 1, preparing the control drug; Step 2, preparing guava glycoside drug solution; Step 3: Use cecal ligation and puncture (CLP) to establish a septic lung injury model.

6. The method for preparing a control drug for treating septic lung injury according to claim 5, characterized in that: The control drug is prepared by mixing dimethyl sulfoxide and corn oil in a ratio of 1 to 9 to obtain the control drug.

7. The method for preparing a drug for treating sepsis-induced lung injury according to claim 5 is characterized in that: The preparation of guajarin comprises first dissolving 10 mg guajarin in 100 ul of dimethyl sulfoxide, then adding 900 ul of sterile corn oil, vortex stirring and mixing by a stirrer, and finally preparing a drug solution with a guajarin concentration of 10 mg / ml.

8. A drug for treating sepsis-induced lung injury to establish a sepsis-induced lung injury model, characterized in that: The cecal ligation and puncture method (CLP) was used to establish a septic lung injury model. Mice aged 6-8 weeks and weighing 20-22g were taken, and 200ul of 5% chloral hydrate was injected into the mouse's abdominal cavity. After the anesthesia took effect, the mouse's abdomen was disinfected with 75% ethanol, and the skin was lifted with sterile ophthalmic forceps. The skin and peritoneum were cut in sequence on the left side of the mouse's mid-abdomen with sterile ophthalmic scissors. The wound was about 0.5cm long. The mouse's cecum was lifted with ophthalmic forceps, and the feces in the cecum were squeezed into the distal end with forceps. The cecum was ligated with No. 4 surgical suture at 2 / 3 of the cecum. The cecum was punctured with an 18-gauge syringe needle and passed out from the opposite side, a small amount of feces was squeezed out, the cecum was sent into the abdominal cavity, the abdomen was sutured, and the skin and peritoneum were cut in sequence on the left side of the mouse's mid-abdomen with sterile ophthalmic scissors. The wound was about 0.5cm long, the mouse's cecum was lifted with ophthalmic forceps, and then the cecum was sent into the abdominal cavity, the abdomen was sutured, and then 1ml was subcutaneously injected into the back of the mouse. Fluid resuscitation was performed with 0.9% NaCI, and mice were observed for death within 2 hours after complete resuscitation.

Images