Application of trigonelline in preparation of medicine for treating sepsis

Abstract

The invention provides application of trigonelline in preparation of a medicine for treating sepsis, and belongs to the technical field of biological medicine. The invention develops a novel medicine, namely the trigonelline for treating sepsis, the treatment effect of the trigonelline on sepsis is detected, and the result proves that the treatment of the trigonelline can inhibit kidney tissue injury caused by sepsis and reduce the expression of inflammatory factors. The natural chemical component trigonelline extracted from plants is used for treating sepsis injury, the safety is high, the treatment effect is good, a new effective treatment medicine is provided for treatment of sepsis, and the problem that an effective sepsis prevention and treatment medicine is lacked clinically is solved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of trigonelline in preparing a drug for treating sepsis. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome and organ dysfunction caused by the invasion of pathogenic microorganisms such as bacteria into the body. It has a high morbidity and mortality rate clinically and has become one of the major diseases that seriously threaten human life. The kidney is one of the organs most commonly affected by organ dysfunction in sepsis. In sepsis, under the action of endotoxins released by bacteria, neutrophils, mononuclear macrophages, vascular endothelial cells, etc. undergo a complex immune network reaction and release a large number of endogenous inflammatory mediators. These inflammatory mediators lead to a decrease in renal perfusion pressure and glomerular filtration pressure, ultimately causing AKI, and acute kidney injury (AKI) has been proven to be an independent risk factor for increased mortality in patients with sepsis. However, the pathogenesis of sepsis AKI is very complex and has not yet been fully elucidated; and there is currently a lack of effective preventive and therapeutic drugs for sepsis AKI in clinical practice. A safe and effective new drug for the treatment of sepsis is urgently needed.

[0003] Trigonelline (Tg) is a plant alkaloid and the main alkaloid component of the leguminous plant fenugreek. Trigonelline has anti-thrombotic, anti-diabetic, antioxidant, anti-inflammatory and neuroprotective effects and can be used as a nutritional additive, pharmaceutical intermediate, etc. At present, there is no research on the use of trigonelline in the treatment of septic injury. Summary of the invention

[0004] The purpose of the present invention is to provide the use of trigonelline in the preparation of a drug for treating sepsis, wherein trigonelline can slow down kidney tissue damage caused by sepsis and inhibit the expression of inflammatory factors.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides application of trigonelline in preparing a medicine for treating sepsis.

[0007] Furthermore, the structure of trigonelline is shown in formula (I):

[0008]

[0009] Furthermore, the drug for treating sepsis may be added with a pharmaceutically acceptable carrier.

[0010] Furthermore, the dosage form of the drug for treating sepsis is one of powder, solution, capsule, granule, tablet, emulsion and suspension.

[0011] The beneficial effects of the present invention compared with the prior art are:

[0012] (1) The present invention has developed a new drug for treating sepsis - trigonelline, and tested the therapeutic effect of trigonelline on sepsis. The results show that trigonelline treatment can treat sepsis, inhibit renal tissue damage caused by sepsis, and reduce the expression of inflammatory factors.

[0013] (2) The present invention utilizes trigonelline, a natural chemical component extracted from plants, to treat septic injury, which has high safety and good therapeutic effect, provides a new effective therapeutic drug for the treatment of sepsis, and improves the problem of lack of effective preventive and therapeutic drugs for sepsis in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0015] Figure 1 This is a result diagram showing that trigonelline improves the survival rate of CLP-induced septic mice in Example 1 of the present invention;

[0016] Figure 2 The figure is a result diagram of the effect of fenugreek on reducing the morphological damage of renal tissue and the expression of inflammatory factors in mice with sepsis induced by CLP in Example 2 of the present invention, wherein A is a HE staining diagram of the morphological changes of renal tissue in each group, and the scale of the picture is 100 μm; B is a quantitative scoring result of renal tissue damage in each group; C is the expression level of inflammatory factor IL-1β mRNA in renal tissue of each group; D is the expression level of inflammatory factor IL-6 mRNA in renal tissue of each group;

[0017] Figure 3 This is a result diagram of Example 3 of the present invention showing that trigonelline alleviates renal injury in septic mice induced by CLP, wherein A is a result diagram of the BUN content in the serum of each group; and B is a result diagram of the Creatinine content in the serum of each group. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Example 1

[0024] Example 1 of the present invention detected the effect of trigonelline on the survival rate of CLP-induced septic mice, and the specific steps were as follows:

[0025] (1) Experimental Materials

[0026] A. Experimental animals: 60 male C57BL / 6J mice aged 6-8 weeks, weighing 16-22 g, were purchased from the Animal Center of Nanfang Hospital and raised in the SPF-grade Animal Experimental Department of Nanfang Hospital, Southern Medical University. All operations involved in the animal breeding process were approved by the Ethics Committee and met the requirements of animal ethics.

[0027] B. Reagents and instruments: Trigonelline (MedChemExpress, USA); isoflurane (Reward Life Science Co., Ltd.); microvascular artery clamp (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk thread (Ningbo Medical Suture Co., Ltd.); physiological saline (Shijiazhuang Siyao Co., Ltd.); phosphate buffer saline (PBS) pH 7.4 buffer (Gibco).

[0028] (2) Animal experiments

[0029] 1) Establishment of the mouse cecal ligation and puncture (CLP) model: The mice were anesthetized by isoflurane breathing. After the mice were anesthetized, the mice were fixed, the abdominal surgical area was shaved and the skin was prepared, and the abdominal skin of the mice was disinfected with 75% alcohol. Then, 1-2 cm was cut along the midline of the abdomen, the epidermis and muscle layer were separated, the cecum was found, and the cecum was ligated with 4-0 surgical absorbable sutures at 3 / 4 of the distance from the end. After ligation, a 1.2*38TWLB needle was used to pierce the blind end of the cecum, and the needle was used to penetrate the cecum. A small amount of intestinal contents were squeezed out from the needle hole of the cecum, and the cecum was returned, and the intestinal contents were avoided from adhering to the surgical incision as much as possible. The muscle layer and skin were sutured layer by layer. After surgery, each mouse was subcutaneously injected with 1 ml of 37℃ preheated physiological saline for fluid resuscitation. 12 hours after surgery, the mice were anesthetized, and then blood was collected from the heart of the mice and kidney tissue was taken for examination. The sham operation group (Sham) underwent only laparotomy without cecal ligation and puncture.

[0030] 2) Experimental groups:

[0031] Sixty C57BL / 6J mice aged 6-8 weeks were randomly divided into Sham group, CLP group and CLP group pretreated with trigonelline (CLP+Tg).

[0032] 1) Sham group: After 1 h pretreatment with intraperitoneal injection of PBS solution, only laparotomy was performed without CLP experiment;

[0033] 2) CLP group: CLP model was established after intraperitoneal injection of PBS solution for 1 h pretreatment;

[0034] 3) CLP+Tg group: One hour after intraperitoneal injection of 10 mg / kg trigonelline solution, the CLP model was established.

[0035] 3) Survival rate statistics

[0036] Observe and record the death time of mice in the above groups, and count the survival time of mice in each group. The results are as follows: Figure 1 shown.

[0037] Figure 1 The results showed that treatment with trigonelline could significantly prolong the survival time of septic mice and increase the survival rate of mice.

[0038] Example 2

[0039] Example 2 of the present invention detected the effects of trigonelline on the morphological damage of renal tissue and the expression of inflammatory factors in CLP-induced septic mice. Figure 2 As shown, the specific method is as follows:

[0040] (1) Experimental Materials

[0041] Experimental Animals:

[0042] Twenty-four male C57BL / 6J mice aged 6-8 weeks and weighing 18-22 g were selected for the experiment. They were purchased from the Animal Center of Nanfang Hospital of Southern Medical University and raised in the SPF-level Animal Experiment Department of Nanfang Hospital of Southern Medical University. All operations involved in the animal breeding process were approved by the Ethics Committee and met the requirements of animal ethics.

[0043] Reagents and instruments

[0044] Trigonelline (MedChemExpress, USA); isoflurane (Reward Life Science Co., Ltd.); microvascular artery clamp (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk thread (Ningbo Medical Suture Co., Ltd.); physiological saline (Shijiazhuang Siyao Co., Ltd.); phosphate buffer saline (PBS) pH 7.4 buffer (Gibco); hematoxylin-eosin staining (Beijing Regen Biological Company); anhydrous ethanol (Guangdong Guanghua Science and Technology Co., Ltd.); xylene (Guangdong Guanghua Science and Technology Co., Ltd.); paraffin (Leica); 4% paraformaldehyde (Beijing Solebold Technology Co., Ltd.); neutral gum (Solarbio); fully automated fluorescence microscope (Olympus); TRIZOL lysis buffer (Invitrogen); chloroform (Guangdong Guanghua); isopropanol (Guangdong Guanghua); anhydrous ethanol (Guangdong Guanghua); DEPC water (Sigma); SYBRGreen fluorescent dye (Toyobo); ReverTraAce qPCR RT Kit (Toyobo); PCR instrument (Eppendorf, Germany); Fluorescence quantitative PCR instrument (Applied Biosystems AB, USA).

[0045] (2) Experimental methods and results

[0046] 1) Animal experiments

[0047] A. Establishment of mouse cecal ligation and puncture (CLP) model: using Example 1.

[0048] B. Experimental Grouping

[0049] Twenty-four C57BL / 6J mice aged 6-8 weeks were randomly divided into Sham group, CLP+PBS group, and CLP group pretreated with trigonelline (CLP+Tg).

[0050] a. Sham group: after 1 h pretreatment with intraperitoneal injection of PBS solution, only laparotomy was performed without CLP experiment;

[0051] b. CLP group: CLP model was established after intraperitoneal injection of PBS solution for 1 h pretreatment;

[0052] c. CLP+Tg group: One hour after intraperitoneal injection of 10 mg / kg trigonelline solution, the CLP model was established.

[0053] 2) Detection of pathological morphological changes in renal tissue

[0054] Fresh renal tissue was immersed in 4% paraformaldehyde for 24 hours, then dehydrated, embedded, sliced, and then stained with hematoxylin-eosin and sealed with neutral gum. The pathological morphological changes of intestinal tissue were observed under an automatic fluorescence microscope.

[0055] 3) Inflammatory factor mRNA expression

[0056] A. RNA extraction method:

[0057] Take 20-50mg of kidney tissue and place it in a 1.5mL RNase-free EP tube. Add 500μL TRIZOL lysis buffer and homogenize. Add 100μL chloroform to the homogenate, shake 15-20 times, and let stand at room temperature for 1-2min. Centrifuge at 12,000rpm, 4℃ for 15min, and pipette the upper aqueous phase into a new 1.5mL RNase-free EP tube. Add an equal volume of isopropanol, shake 15-20 times, and let stand at room temperature for 10min. Centrifuge at 12,000rpm, 4℃ for 10min, discard the supernatant, add 1mL 80% ethanol (prepared with DEPC water), shake and oscillate, and centrifuge at 7500rpm, 4℃ for 5min. Discard the supernatant, centrifuge at 7500rpm, 4℃ for 1min, absorb the residual ethanol, let stand at room temperature for 5-10min, and add 50-100μL DEPC water to resuspend. The RNA product is stored in a -80℃ refrigerator for later use.

[0058] B. Reverse transcription of extracted RNA:

[0059] Take 1μL of extracted RNA and add it to 6μL of NucLease-freeWater. Denature it at 65℃ for 5min on a PCR amplifier. Immediately take it out and cool it on ice after denaturation. Add 2μL of 5×reaction buffer, 0.5μL of RT Enzymemix, and 0.5μL of Prime mix to the reaction system. Slightly shake it and centrifuge it for 3-5s. Place the reaction system on a PCR amplifier, reverse transcribe it at 37℃ for 15min, and inactivate the enzyme at 98℃ for 5min. After the reaction, add 190μL of sterile water to obtain a cDNA solution.

[0060] C. Read-time PCR reaction:

[0061] 6 μL SYBR Green, 1 μL inflammatory factor primer and 5 μL cDNA solution were added to the reaction system and then placed in a fluorescent quantitative PCR instrument. ReaL-time PCR reaction conditions: CycLing: stage 95℃15s; 60℃ 1min, 40 cycles; MeLt cure stage: 95℃ 15s; 60℃ 1min; 95℃30s; 60℃ 15s.

[0062] Figure 2 A, B The results of HE staining and scoring of renal tissue showed that compared with the Sham group, the renal tubular necrosis and tubular shrinkage of the mice in the CLP model group were significantly increased, and the morphological changes of the renal tissue of the mice were significantly improved after treatment with trigonelline. Figure 2 Data in C and D show that the expression levels of inflammatory factors IL-1p and IL-6 mRNA in renal tissue of the trigonelline group were significantly downregulated compared with CLP. The experimental results indicate that trigonelline can inhibit the pathological morphological damage and expression of inflammatory factors in renal tissue of mice induced by CLP.

[0063] Example 3

[0064] Example 3 of the present invention detected the therapeutic effect of trigonelline on renal tissue damage in CLP-induced septic mice, and the specific steps were as follows:

[0065] (1) Experimental Materials

[0066] Experimental animals

[0067] Twenty-four male C57BL / 6J mice aged 6-8 weeks and weighing 18-22 g were selected for the experiment. They were purchased from the Animal Center of Nanfang Hospital of Southern Medical University and raised in the SPF-level Animal Experiment Department of Nanfang Hospital of Southern Medical University. All operations involved in the animal breeding process were approved by the Ethics Committee and met the requirements of animal ethics.

[0068] Reagents and instruments

[0069] Trigonelline (MedChemExpress, USA); isoflurane (Reward Life Science Co., Ltd.); microvascular artery clamp (Chengdu North America Jiarui Biotechnology Co., Ltd.); sterile silk thread (Ningbo Medical Suture Co., Ltd.); normal saline (Shijiazhuang Siyao Co., Ltd.); phosphate buffered saline (PBS) pH 7.4 buffer (Gibco); blood urea nitrogen (BUN) detection kit (Nanjing Jiancheng Bioengineering Institute); blood creatinine (Creatinine) detection kit (Nanjing Jiancheng Bioengineering Institute).

[0070] (2) Experimental methods and results

[0071] 1) Animal experiments

[0072] Establishment of mouse cecal ligation and puncture (CLP) model: using Example 1.

[0073] 2) Experimental Grouping

[0074] Twenty-four C57BL / 6J mice aged 6-8 weeks were randomly divided into Sham group, CLP+PBS group, and CLP group pretreated with trigonelline (CLP+Tg).

[0075] Sham group: After 1 h pretreatment with intraperitoneal injection of PBS solution, only laparotomy was performed without CLP experiment;

[0076] CLP group: CLP model was established after 1 h pretreatment with intraperitoneal injection of PBS solution;

[0077] CLP+Tg group: One hour after intraperitoneal injection of 10 mg / kg trigonelline solution, the CLP model was established.

[0078] 3) Detection of BUN and Creatinine levels in serum

[0079] The serum was collected from the heart of the dissected mouse and centrifuged. The BUN and Creatinine detection kit for mouse serum was tested according to the instructions. The results were as follows: Figure 3 shown.

[0080] Figure 3 A, B Serum BUN and Creatinine levels showed that compared with the Sham group, the serum BUN and Creatinine levels of mice in the CLP group increased significantly; compared with the CLP group, the serum BUN and Creatinine levels were significantly reduced after pretreatment with trigonelline. The above experimental results indicate that trigonelline can alleviate CLP-induced septic renal injury in mice.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of trigonelline in the preparation of drugs for treating sepsis.

2. The use of trigonelline in the preparation of a drug for treating sepsis according to claim 1, characterized in that: The structure of the trigonelline is shown in formula (I):

3. The use of trigonelline in the preparation of a drug for treating sepsis according to claim 2, characterized in that: The drug for treating sepsis may be added with a pharmaceutically acceptable carrier.

4. The use of trigonelline in the preparation of a drug for treating sepsis according to claim 3, characterized in that: The dosage form of the drug for treating sepsis is one of powder, solution, capsule, granule, tablet, emulsion and suspension.

Images