CN111789859A - Application of dexamethasone in the preparation of medicines for preventing or relieving jellyfish stings - Google Patents

Abstract

The invention relates to the technical field of medicine, and provides the application of dexamethasone or its derivatives in the preparation of medicines for preventing or relieving jellyfish stings. It was verified by experiments that the administration of mice 10 minutes before the sting can significantly prolong the survival time of mice and reduce the mortality of mice; significantly reduce the changes of cardiac function indexes, liver function indexes and kidney function indexes of mice caused by jellyfish toxin; Transcriptomic analysis of mice found that administration of 10 minutes before sting in mice can significantly improve the combined damage of multiple organs, reduce the degree of inflammation between multiple organs, and prevent jellyfish stings for dexamethasone or its derivatives. provides a new theoretical basis. Because dexamethasone is a mature anti-inflammatory and immunosuppressive agent, its trade name includes dexamethasone acetate tablets, etc., its pharmacological action is clear, the toxic and side effects are small, and the drug safety has been clinically recognized. New indications can quickly achieve clinical translation.

Description

Application of dexamethasone in the preparation of medicines for preventing or relieving jellyfish stings

technical field

The invention belongs to the field of biomedicine and provides a new use of dexamethasone, in particular to the application of dexamethasone or a derivative thereof in preparing a medicine for preventing or relieving jellyfish stings.

Background technique

Jellyfish is one of the oldest and mysterious creatures in the world. It has existed on the earth about 650 million years ago and is distributed in almost all seas. In terms of numbers, the number of jellyfish has exploded in recent decades, not only leading to the destruction of marine ecosystems and causing catastrophic damage to marine fisheries, but also increasing jellyfish stings, with thousands of people injured every year , becomes a very difficult problem.

Since the 1940s, Pubmed has been following and gradually covering the Portuguese man-of-war, Irukandji, Chironex fleckeri, Stomolophus meleagris, and Cyanea capillata ) and other representative cases of poisonous jellyfish stings, treatment and related research on poisoning mechanism, it is basically clear that jellyfish stings can be divided into two categories: local skin symptoms and systemic poisoning. Local skin symptoms include severe pain, itching, rash, pigmentation, etc.; systemic poisoning symptoms are caused by severe inflammatory reactions after jellyfish stings. If soaked in jellyfish toxins for a long time, jellyfish toxins are likely to penetrate the skin and enter blood, further causing extensive damage to multiple organs in the body such as the heart, liver, and kidneys, resulting in death.

At present, the mainstream view holds that cardiovascular toxicity is the main cause of death of jellyfish toxin (TE). Antitoxin, MgSO ₄ , and Ca ²⁺ channel blockers are effective therapeutic agents for jellyfish delayed toxicity systemic syndrome (DJES). However, there are significant differences in the antagonistic effects between different jellyfish, and even the intervention effects of the same experiment in different laboratories will be completely opposite, such as C. fleckeri antitoxin, MgSO ₄ and verapamil.

Dexamethasone (DXMS) is an artificial glucocorticoid with significant anti-inflammatory and immunosuppressive effects. It is widely used clinically in asthma, allergy and other diseases, and exerts its physiological effects through the glucocorticoid receptor (GR). However, the preventive effect of dexamethasone in maternal stings has not been reported in the literature.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new medicinal use of dexamethasone or its derivatives.

The first aspect of the present invention is to provide the use of dexamethasone or its derivatives in the preparation of a medicine for preventing or alleviating jellyfish stings. reaction to the drug.

Preferably, the drug for reducing the changes of blood indexes after sting is a drug for reducing the changes of blood indexes of heart function, liver function and renal function.

After the application of dexamethasone, the changes of cardiac function, liver function and renal function blood indexes were much lower than those of the PBS-TE group as a positive control, such as the blood indexes of cardiac function, lactate dehydrogenase (LDH), creatine kinase (CK). ), the level of creatine kinase isoenzyme (CK-MB) was significantly lower than that in the PBS-TE group. In terms of liver function blood indicators, the levels of alanine transferase (ALT), aspartate transferase (AST) and total bilirubin (TBIL), direct bilirubin (DBIL) and indirect bilirubin (IBIL) were significantly Lower than PBS-TE group; total protein (TP), albumin (ALB), globulin (GLB) levels were significantly higher than PBS-TE group. In terms of renal function blood indexes, the level of creatinine (Cr) was slightly higher than that of the positive control group, and the level of urea nitrogen (BUN) was significantly lower than that of the positive control group. However, the above blood indexes were all different from those in the blank control group, indicating that DXMS could significantly inhibit and improve the symptoms of DJES, but could not completely reverse the symptoms.

Preferably, the drug for reducing the inflammatory response after a sting is a drug for inhibiting the NF-κB signaling pathway. The NF-kB signaling pathway may play an important role in the systemic poisoning of jellyfish stings and become a potential new mechanism for systemic poisoning of jellyfish stings.

The dexamethasone derivatives in the present invention refer to dexamethasone compounds capable of exerting pharmacological effects, such as hydrocortisone, prednisone and the like.

In order to verify the protective effect of dexamethasone on multi-organ damage caused by jellyfish stings, the present invention uses mouse survival experiments, hematology experiments, multi-organ slices and multi-organ transcriptomics to verify that dexamethasone is effective in jellyfish. Protection against multiple organ damage from stings.

ICR mice were selected, 7-week-old male mice were used for the experiment, and dexamethasone (3 mg/kg) was injected into the tail vein of mice 10 minutes before the sting. Mice were stinged by tail vein injection of jellyfish toxin at a dose of 2 mg/kg, and the mice were placed in a 1000 ml lunch box containing 0.5 cm high sea water.

Through experiments, it was found that the administration of mice 10 minutes before the sting can significantly prolong the survival time of mice; significantly reduce the changes of cardiac function indexes, liver function indexes and kidney function indexes of mice caused by jellyfish toxin; Transcriptomic analysis showed that drug administration 10 minutes before sting in mice could significantly improve the joint injury of multiple organs and attenuate the degree of inflammation between multiple organs.

The medicine for preventing or relieving jellyfish stings of the present invention is dexamethasone or its derivatives as the only active ingredient or a pharmaceutical composition comprising dexamethasone or its derivatives.

The medicament or pharmaceutical composition of the present invention can be made into any dosage form with commonly used pharmacy auxiliary materials, for example, it can be decoction, powder, pill, wine, lozenge, glue, tea, koji, cake Dosage, lotion, stick, thread, strip, nail, moxibustion iron, ointment, elixir, liposome preparation, aerosol, injection, mixture, oral ampoule, tablet, capsule, drop Pills, emulsions, films or sponges.

The above-mentioned medicine or pharmaceutical composition of the present invention is suitable for marine workers, navy soldiers, seaside tourists and marine lifeguards who encounter jellyfish stings, and can be used for preventing or alleviating jellyfish stings, that is, it is administered in advance to those who may encounter jellyfish stings. The population of the drug is not limited to oral administration, injection, etc.

The role and effect of the invention

It was verified by experiments that the administration of mice 10 minutes before the sting can significantly prolong the survival time of mice and reduce the mortality of mice; it can significantly reduce the changes of cardiac function indexes, liver function indexes and kidney function indexes of mice caused by jellyfish toxin; Transcriptomic analysis of mice found that administration of 10 minutes before sting in mice can significantly improve the combined damage of multiple organs, reduce the degree of inflammation between multiple organs, and prevent jellyfish stings for dexamethasone or its derivatives. provides a new theoretical basis.

In addition, since dexamethasone is a mature anti-inflammatory and immunosuppressive agent, its trade name includes dexamethasone acetate tablets, etc., its pharmacological action is clear, the toxic and side effects are small, and the drug safety has been clinically recognized. The new indication of metasone can achieve clinical translation sooner. In addition, dexamethasone's target "NF-kB channel" has little difference in expression in different tissues (currently reported to be mainly highly expressed in liver, kidney, muscle, vascular endothelium and other tissues), compared with other anti-inflammatory, specificity Weak, and jellyfish toxin is a mixed compound, and specific anti-inflammatory drugs are not suitable. Therefore, dexamethasone has great potential in the protection application of multi-organ damage caused by jellyfish stings, and the present invention also provides a new clinical medicine for preventing the damage caused by jellyfish stings.

Description of drawings

Figure 1 is a graph showing the effect of dexamethasone on the survival of stinged mice, wherein A is the effect of different doses of dexamethasone on the survival rate of mice within 24 hours, and B is the result of the half effective dose of dexamethasone. Used to measure the survival of mice.

Figure 2 is a graph showing the results of the hematological effects of dexamethasone on sting-induced mice. Among them, A is the change level of lactate dehydrogenase (LDH), creatine kinase (CK), and creatine kinase isoenzyme (CK-MB) in injury-related enzyme indicators Contrast; B is the comparison of changes in alanine aminotransferase (ALT) and aspartate transferase (AST) in damage-related enzyme indexes; C is total bilirubin (TBIL) ), direct bilirubin (DBIL), and indirect bilirubin (IBIL) level comparison; D is total protein (TP), albumin (ALB), globulin (globulin) , GLB) change level comparison; E is the change level comparison of creatinine (creatinine, Cr); F is the change level comparison of urea nitrogen (BUN).

Figure 3 Comparison of changes in organ signaling pathways in mice after pre-injection of dexamethasone: A is a comparison of changes in the differential signaling pathways of the heart; B is a comparison of changes in the differential signaling pathways of the liver; C is a comparison of changes in the differential signaling pathways of the kidneys .

Figure 4 is a graph showing the changes of inflammatory factors in various organs of mice after dexamethasone was stinged.

Detailed ways

The present invention will be described in detail below with reference to the embodiments and accompanying drawings. However, the following examples should not be construed as limiting the scope of the present invention.

Example 1: Dexamethasone can improve the survival and mortality of mice after sting

1. Experimental method

Male mice aged 7 weeks of ICR were selected for the experiment, and the experimental mice were divided into four groups: experimental group DXMS-TE group (n=12), positive control group PBS-TE group (n=12), negative control group DXMS -PBS group (n=12), blank control group PBS-PBS group (n=12).

After weighing and recording the weight of the mice, the first corresponding reagent of each group was injected intravenously according to the dose of 0.5ml/10mg, and 10 minutes later, the second corresponding reagent of each group was injected again according to the dose of 0.5ml/10mg. Each mouse was injected twice in succession, two mice in each group were injected in turn, put into two perforated lunch boxes containing 20 ml of seawater, and the injection time was recorded.

The four groups should be tested in parallel, and the above steps should be repeated six times. After TE injection, the mice were continuously observed for 24 hours, and the poisoning symptoms of the mice were observed, and the death time and number of the mice were recorded, and the median survival time and survival curve of the mice were calculated using GraphPadprism 7.00 software.

2. Experimental results

The results showed that all the mice in the positive control group had poisoning symptoms such as lying down, trembling, and staggering, and the DJES mouse model was successfully constructed. Although the mice in the experimental group also showed symptoms of poisoning, the degree of poisoning was significantly lighter than that of the mice in the positive control group.

We used the method of injecting dexamethasone into the tail vein of DJES model mice to analyze the survival time in the toxicological evaluation, and found that injection of high doses of DXMS can significantly increase the mortality of mice and improve the survival of mice. (No obvious lying down, trembling, staggering and other symptoms).

The median time to death in mice was inversely proportional to the DXMS concentration. The performance of DJES mice injected with DXMS in advance can be divided into: >3mg/kg, survival rate >75%, with obvious antagonistic effect; 1-3mg/kg, survival rate of 50%-75%, less antagonistic effect; ~1mg/kg, almost no antagonistic effect, the survival rate was 15% to 25% (Figure 1A); after more than 24h, all mice began to eat and drink normally, and gradually recovered, no death phenomenon occurred. Finally, the half effective dose of DXMS (IC50)=1.005 mg/kg was obtained by measuring the corresponding survival rate under the gradient concentration (Fig. 1B).

Example 2: Dexamethasone can improve the changes of hematological indexes in mice

1. Experimental method:

In order to verify the effect of dexamethasone on blood indexes of multiple organs after sting, blood biochemical analysis was performed on the blood of mice after sting.

Male mice aged 7 weeks of ICR were selected for the experiment, and the experimental mice were divided into four groups: experimental group DXMS-TE group (n=6), positive control group PBS-TE group (n=6), negative control group DXMS -PBS group (n=6), blank control group PBS-PBS group (n=6). 8 hours after the injection of the reagents in each group of mice, the orbital blood was collected into a centrifuge tube soaked in 0.5% heparin sodium solution, centrifuged at 3000 r for 10 min, the supernatant was collected, and the following indicators were measured immediately using the SIEMENS kit:

Liver and kidney function related indicators: total protein (total protein, TP), albumin (albumin, ALB), globulin (globulin, GLB), total bilirubin (total bilirubin, TBIL), direct bilirubin (direct bilirubin, DBIL), indirect bilirubin (IBIL), creatinine (Cr), urea nitrogen (BUN); damage-related enzyme indicators: alanine aminotransferase (ALT), aspartate transfer Enzyme (Aspartate transferase, AST), lactate dehydrogenase (lactate dehydrogenase, LDH), creatine kinase (creatine kinase, CK), creatine kinase isoenzyme (creatine kinase isoenzyme, CK-MB).

2. Experimental results

Among the cardiac function indexes, the mice in the PBS-TE group were significantly higher than those in the blank control group; the LDH, CK and CK-MB in the DMXS-TE group were significantly lower than those in the PBS-TE group, of which CK and CK-MB had specific cardiac function. The functional index of DXMS-TE mice was significantly lower than that of PBS-TE group (Fig. 2A).

Among the liver function indexes, ALT, AST, TBIL, DBIL, IBIL in the DXMS-TE group were significantly lower than those in the PBS-TE group, and only slightly higher than those in the PBS-PBS group as a blank control; while the PBS-TE group was significantly higher than the blank control group. control group (Figure 2B and Figure 2C). For TP, ALB and GLB, the PBS-TE group was significantly lower than the blank control group, where TP was significantly lower, while DXMS-PBS was significantly higher than PBS-TE, and GLB was significantly higher (Fig. 2D).

Among the renal function indexes, the sCre of the DXMS-TE group was slightly higher than that of the positive control group, while the positive control group was slightly higher than the blank control group. Interestingly, the negative control group was significantly lower than that of the blank control group (Figure 2E). The DXMS-TE group was significantly lower than the positive control group, but higher than the blank control group, and the positive control group was significantly higher than for the blank control group and the negative control group (Fig. 2F).

The above results indicated that DXMS could significantly inhibit and improve the symptoms of poisoning in mice, but could not completely reverse the symptoms.

Example 3: Dexamethasone can significantly improve the increase of inflammatory indexes in multiple organs in mice

1. Experimental method:

Experiments were divided into four groups: DXMS-TE group (n=4), DJES group (n=4), negative control group (n=4) and blank control group (n=4). Kidney, liver, heart, and skin samples from each group of mice were taken 8 h after reagent injection, followed by total RNA extraction, rRNA removal by OligodT enrichment, strand-specific cDNA library construction, and Illumina HiSeq4000 sequencing. The analysis work after the computer data includes the FastQC software to evaluate the sequencing quality, the Hisat2 software to compare the reference genome, the StringTie software to estimate the transcript abundance, the R software Ballgown to calculate the FPKM and the screening of differential genes, etc.; further PCA analysis, correlation analysis, differential genes Cluster analysis, GO functional annotation and KEGG functional annotation were completed by Kangcheng Bio's custom program Python/R/Shell.

2. Experimental results

Pathological sections and hematological indicators found that the damage symptoms of multiple organs in the experimental group were significantly weakened compared with the positive control group, but there was still weak damage compared with the blank control group. Therefore, we used the method of multi-organ transcriptome sequencing to detect the four groups. Differences in the expression of genes in important organs in the mouse model and analysis of their potential mechanisms of action.

We constructed a total of 48 transcriptomes of samples, including blank control, positive control, negative control, and pre-injected DXMS for the DJES model. n=4.

After DXMS-TE and positive control analysis, 29178, 26675 and 29842 differentially expressed genes were detected in the heart, liver and kidney organs, respectively. Among them, 13,505 differentially expressed genes were up-regulated and 15,673 were down-regulated in the heart; 14,480 genes were up-regulated and 12,195 genes were down-regulated in the liver; 19,953 genes were up-regulated and 9,889 genes were down-regulated in the kidney. a gene. After comparing the experimental group with the blank control, 28,517, 26,710 and 30,314 differentially expressed genes were detected in the heart, liver and kidney organs, respectively, of which 13,409 were up-regulated in the heart and 15,408 were down-regulated. In the kidney, 12,685 genes were up-regulated and 14,025 genes were down-regulated; 14,657 genes were up-regulated and 15,657 genes were down-regulated in kidney.

Then we used the automatic annotation system KAAS (KEGG Automatic Annotation Server) to annotate the differentially expressed genes in 3 organs: compared with the positive control group, the differentially expressed genes in the heart were annotated to 297 signaling pathways, and the differentially expressed genes in the liver were annotated to 297 signaling pathways. The genes were annotated to 313 signaling pathways, and the differentially expressed genes in the kidney were annotated to 302 signaling pathways. According to the enrichment degree of up-regulated genes, the top 15 differentially enriched signaling pathways, such as TNF- signaling-pathway, IL-17-signaling-pathway, Toll-like-receptor-signaling-pathway, NF-kappa-B-signaling-pathway, etc. and found that almost all are related to inflammation, immunity and stress.

At the same time, differential gene analysis was performed between the experimental group and the positive control group and the blank control group. In the previous experiment, 33 multi-organ co-upregulated inflammation-related genes were detected in the DJES model group. Differential expression of each inflammatory factor in the heart, liver and kidney of mice. It was found that the inflammatory factors in the experimental group and the positive control group were generally up-regulated, and the positive control group was significantly up-regulated, while most of the up-regulation in the experimental group was significantly lower than that in the positive control group, especially Tlr2 and Pde4B molecules also appeared down-regulated. (-0.21 for Tlr2 in kidney, -0.01 and -0.29 for Pde4B in heart and kidney). However, there are still several inflammatory factors that are almost the same as the positive control group (difference fold difference <1), S100a8, S100a9, Adamts4, Junb and Egr1 in the heart, S100a9 expression fold exceeds that of the positive control group, and Ccl5, S100a8 in the kidney , S100a9, and Junb, and Cxcl1, Cxcl9, Serpina3f, and Junb in the liver, where the expression fold of Cxcl9 and Serpina3f exceeded that of the positive control group (Fig. 4).

The differential gene analysis between the experimental group and the positive control group was further carried out, and it was found that in the organs of mice injected with DXMS, 33 inflammatory factors were significantly down-regulated, and only a few inflammatory factors were slightly up-regulated. were 0.03 and 0.10, and the fold differences between Cxcl9 and Serpina3f in liver were 0.06 and 0.15. It is suggested that pre-injection of DXMS can significantly antagonize and improve the severe inflammation, immune or stress response in the DJES model, and significantly improve the survival rate and survival status of mice, revealing that the inflammatory response plays an important role in the systemic poisoning of jellyfish stings. damage effect. Through mechanism analysis, DXMS exerts an anti-inflammatory effect by inhibiting the expression of NF-kB signaling pathway. The NF-kB signaling pathway may play an important role in the systemic poisoning of jellyfish stings and become a potential new mechanism for systemic poisoning of jellyfish stings. .

The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principle of the present invention. Such changes and improvements fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

1. Application of dexamethasone or its derivatives in the preparation of a medicament for preventing or relieving jellyfish stings. 2. the application of dexamethasone or its derivative according to claim 1 in the preparation prevention or alleviation of jellyfish sting medicine, it is characterized in that: Wherein, the medicine for preventing or alleviating jellyfish stings is a medicine for reducing the changes of blood indexes after stings or reducing the inflammatory response after stings. 3. the application of dexamethasone or its derivative in preparation prevention or alleviation jellyfish sting medicine according to claim 2, is characterized in that: Wherein, the medicine for reducing the change of blood index after sting is a medicine for reducing the change of blood index of heart function, liver function and kidney function. 4. the application of dexamethasone or its derivative in preparation prevention or alleviation jellyfish sting medicine according to claim 2, is characterized in that: Wherein, the drug for reducing inflammatory response after sting is a drug for inhibiting NF-κB signaling pathway. 5. The application of dexamethasone or its derivative according to any one of claims 1 to 4 in the preparation of a medicine for preventing or relieving jellyfish stings, characterized in that: Wherein, the drug for preventing or alleviating jellyfish stings is dexamethasone or its derivatives as the only active ingredient or a pharmaceutical composition comprising dexamethasone or its derivatives. 6. the application of dexamethasone or its derivative according to claim 1 in the preparation prevention or alleviation of jellyfish sting medicine, it is characterized in that: Wherein, the medicine for preventing or alleviating jellyfish stings is decoction, powder, pill, wine, lozenge, glue, tea, koji, cake, dew, stick, thread, strip, nail medicine, moxibustion iron, ointment, elixir, liposome preparation, aerosol, injection, mixture, oral ampoule, tablet, capsule, drop pill, emulsion, film or sponge.

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