CN110585192A

CN110585192A - Application of loganin in preparation of medicine for treating acute enteritis

Info

Publication number: CN110585192A
Application number: CN201910969300.3A
Authority: CN
Inventors: 宋刚
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2019-12-20

Abstract

An application of loganin in preparing a medicine for treating acute enteritis relates to a new medical application of loganin. In vitro and in vivo experiments prove that loganin has no obvious toxic or side effect on cells and mice, can obviously and effectively treat DSS-induced acute enteritis of mice, can obviously interfere LPS-induced acute inflammation of colon cancer cells, and inhibits the expression of proinflammatory factors. The loganin has safe and effective effect in preparing the medicine for treating acute enteritis, is superior to the traditional medicine, thereby widening the application range and providing a new way for treating acute enteritis.

Description

Application of loganin in preparation of medicine for treating acute enteritis

Technical Field

The invention relates to a new medical application of loganin, in particular to an application of loganin in preparing a medicine for treating acute enteritis.

Background

Loganin, also called Loganin, is an iridoid glycoside compound with highest content in Corni fructus, and has molecular formula of C₁₇H₂₆O₁₀The structural formula is as follows:

loganin is used as the main active ingredient of dogwood and many compounds, and has obvious effects of resisting inflammation, regulating immunity and strengthening heart. Currently, there are research results showing that the main pharmacological activities of loganin include:

1) anti-shock: researches show that loganin has the effects of slowly increasing and maintaining blood pressure, improving heart rate, improving renal blood flow, improving survival rate of shock animals and the like, and has more obvious effect when being combined with synephrine.

2) And (3) immune regulation: loganin is an immunosuppressant, but can regulate immune response in two directions due to different concentrations. In vivo and in vitro experiments show that loganin with proper concentration can promote lymphocyte transformation, IL-2 production and lymphocyte activated killer cell (LAK) proliferation, promote Mixed Lymphocyte Reaction (MLR) of mice and human, and has inhibiting effect at high concentration.

3) Inhibition of preadipocyte differentiation: studies show that loganin 8, loganin 16 and loganin 32 mu m can promote the proliferation of preadipocytes of rats, inhibit the increase of phosphoglycerol dehydrogenase (GPDH) and fat accumulation in the differentiation process of the preadipocytes of the rats, and are obviously in a dose-effect relationship.

4) Neuroprotective effect: studies have shown that loganin, a natural product extracted from the plant Cornus officinalis, can relieve the release of cytochrome C (cytochromeC) through the H-activated protein kinase (MAPK) pathway by up-regulating the ratio of B-lymphocytoma-2 (B-cell lymphoma-2, Bcl-2) and Bcl-2-associated X protein (Bcl-2associated X protein, Bax) and inhibiting the release of cytochrome C (cytochromeC) through the amino-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK) pathways₂O₂Induced oxidative stress of SH-SY5Y cell line in addition, loganin can also save the cell damage of PC12 by activating the nuclear factor- к B (NF-kappa B) pathway.

5) Anti-inflammatory: loganin can increase secretion of glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (BF-GF), and inhibit interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), thereby inhibiting cell injury mediated by inflammatory reaction.

Ulcerative Colitis (UC) is a chronic nonspecific colonic inflammatory disease that is characterized clinically by chronic relapsing, persistent and refractory characteristics. The initiation and the deterioration of UC intestinal inflammation are all started from the injury of intestinal mucosa barrier, the translocation of various antigens in the intestinal tract, the triggering of the immune system of the lamina propria, the induction of the secretion increase of proinflammatory factors, the inhibition of the anti-inflammatory factors and the final induction of the cascade amplification immune inflammatory reaction. The imbalance of proinflammatory-anti-inflammatory factors is an important pathological substance causing intestinal immunoregulation disorder and tissue damage, and is also a core link of UC disease progression. In addition, genetic factors, environmental stimuli and dietary factors interact with immune factors, which results in delayed and serious UC course.

At present, the treatment of UC mainly comprises:

1) traditional therapeutic drugs: amino acid salicylic acid drugs (5-ASA), adrenoglucocorticoid (GCS), immunosuppressant (cyclosporin A), and the like;

2) biological therapeutic agent: inflixis (IFX);

3) a microecological preparation: probiotics, prebiotics, and synbiotics;

4) antibiotics: penicillins, tobramycin, quinolones and cephalosporins;

5) other treatments: traditional Chinese medicine treatment, operation treatment, interventional therapy and the like. Among the treatment means, decoction, powder and the like in traditional Chinese medicine treatment are the most effective, convenient and cheap. However, at present, an effective control means for the chronic evolution of UC still needs to be further explored, and a new traditional Chinese medicine preparation with more targeting and a combined strategy of traditional Chinese medicine and western medicine are hopefully searched.

Disclosure of Invention

The invention aims to provide application of loganin in preparation of a medicine for treating acute enteritis.

The loganin has a molecular formula of C₁₇H₂₆O₁₀The structural formula is as follows:

the loganin can be used for preparing a medicine for treating acute enteritis, wherein the acute enteritis can be acute colitis of mice induced by Dextran Sulfate Sodium Salt (DSS) and acute inflammation of colon cancer cells induced by Lipopolysaccharide (LPS).

The application can be verified using the following experiments:

1. animal experiments, the invention takes C57/BL6 mice as research objects, an acute colitis model is constructed by using 2% DSS, the total duration is 7 days, the treatment with drugs is started on the 3 rd day of the model, the dosage of loganin is 80mg/kg and 160mg/kg, the administration is carried out in a gastric lavage mode, the treatment is continuously carried out for 6 days once a day, in addition, the damage of DSS to colon is weakened by using Sulfasalazine (SASP) as a positive control of loganin, the experiment is ended on the 10 th day, the mice are killed to detect relevant inflammatory indexes, important indexes of acute colitis, namely weight, length of colon, Disease Activity Index (DAI), the loganin is found to weaken the damage of DSS to the colon and the influence on the weight and the length of the colon of the mice, the pathological detection is further found, the loganin can weaken the edema degree of intestinal wall caused by the DSS, the inflammation degree of the intestinal wall and the mucosa deficiency degree, the loss of goblet cells is further weakened, the influence degree of the treatment, the influence of local inflammatory factors on the intestinal wall, the expression mechanism of the inflammatory factors of the loganin, beta-TNF-beta-.

2. In vitro cytology experiments: taking a colon cancer cell HCT116 as a research object, pretreating by using loganin for 16h, adding LPS to induce acute inflammation, collecting samples after 3h, and detecting the expression change of inflammatory factors. In addition, the effect of different concentrations of loganin on the cellular activity of HCT116 was examined. The result shows that loganin can inhibit acute inflammation of colon cancer cells induced by LPS, and has no obvious influence on proliferation of the colon cancer cells.

The invention has the following outstanding technical effects:

1) loganin can effectively treat DSS-induced acute enteritis in mice, and the specific mechanism is related to NF- к B/STAT3 signal pathway.

2) Loganin has no cytotoxicity to colon cancer cells, and can interfere acute inflammation of colon cancer cells induced by LPS and inhibit expression of proinflammatory factors.

3) The loganin has the function of preparing the medicine for treating acute enteritis, is safe and effective, is superior to the traditional medicine, thereby widening the application range and providing a new way for treating acute enteritis.

Drawings

FIG. 1 shows the inflammation schedule of DSS-induced acute enteritis in mice, and the change in body weight of mice during acute enteritis treatment with Loganin (grouped into Control group (Control), model group (DSS), Loganin low concentration group (DSS + Logan (80mg/kg)), Loganin high concentration group (DSS + Logan (160mg/kg)), and SASP positive Control group (DSS + SASP (100 mg/kg)).

Fig. 2 shows the results of statistical analysis of the weight loss on the last day (day nine) of molding and treatment termination, for each group (n-9,^*p<0.05,^***p<0.001,^**p<0.01,^****p<0.0001)。

figure 3 is a statistical analysis of the disease activity index of loganin on inflammation of acute enteritis in mice (n-9,^**p<0.01,^****p<0.0001)。

figure 4 is a general graph of the effect of DSS-induced acute inflammation on colon length and the change in colon length after loganin treatment.

Figure 5 is a statistical analysis of the effect of DSS-induced acute inflammation on colon length and the change in colon length after loganin treatment (n-6,^***p<0.001，^****p<0.0001)。

FIG. 6 is a graph showing the effects of HE in detecting the treatment effect of loganin on colitis (Control group), model group (DSS), loganin low concentration group (DSS + Log (80mg/kg)), loganin high concentration group (DSS + Log (160mg/kg)), and SASP positive Control group (DSS + SASP (100 mg/kg)).

Figure 7 is a statistical analysis of the histological scores for loganin-relieved colitis based on HE results (n-3,^**p<0.01,^***p<0.001)。

FIG. 8 is a graph in which glycogen staining (PAS) was used to examine the effect of loganin on the ability of goblet cells of the intestinal epithelium to secrete polysaccharides (Control, model (DSS), loganin low concentration (DSS + Log (80mg/kg)), loganin high concentration (DSS + Log (160mg/kg)) and SASP positive Control (DSS + SASP (100mg/kg)), with arrows indicating PAS positive staining particles.

Fig. 9 shows the positive statistics for PAS in fig. 8 (n 3, p <0.01, p < 0.001).

Fig. 10 is a graph showing the effect of loganin treatment on inflammatory factor IL-1 β in mice enteritis (control group (ctrl, n ═ 3); (DSS + Log L, n ═ 3); (DSS + Log H, n ═ 3); (DSS + sapp).

FIG. 11 shows the effect of loganin treatment on IL-6, an inflammatory factor in mice enteritis.

FIG. 12 is a graph showing the effect of loganin treatment on the inflammatory factor TNF- α in mice enteritis.

FIG. 13 shows the protein expression regulation of p-p65 and p-STAT3, key proteins of the inflammatory signaling pathway NF- к B/STAT3 by loganin.

Figure 14 is a statistical analysis of the protein expression levels of the key pathway proteins p-p65 and p-stat3 (n-6,^*p<0.05,^***p<0.001,^****p<0.0001)。

FIG. 15 shows MTT assay to determine the effect of loganin at various concentrations (0, 0.5 μm, 1 μm, 5 μm and 12.5 μm) on the proliferative activity of HCT116, colon cancer cells.

FIG. 16 shows the regulation of the expression of inflammatory factors (IL-1. beta., IL-6, TNF-. alpha., and IFN-. gamma.) in LPS-induced acute enteritis by real-time fluorescent quantitative PCR (RT-PCR) detection of loganin in HCT116 cells.

Detailed Description

The following examples will further illustrate the present invention with reference to the accompanying drawings.

Example 1 animal experiments

1. The main experimental materials: experimental animals, C57/BL6 mice (6-8 weeks, male, 18-20g in weight), purchased from Shanghai Si Rick laboratory animals Co., Ltd, SPF grade, were housed in the laboratory animals center, Xiamen university. DSS and SASP, available from Shanghai assist saint Biotech, Inc. Reagents and kits required by experiments are purchased from Cell Signaling company and Nanjing institute of bioengineering.

2. The experimental method comprises the following steps:

(1) mouse colitis model 50 mice were randomly assigned to 5 groups (n-10): a control group, a DSS model group, a low dose loganin group (80mg/kg/d), a high dose loganin group (160mg/kg/d), and a positive control group SASP group (100 mg/kg/d). The method is briefly described as follows, 2% DSS is administered by drinking water for seven consecutive days, beginning on the third day by gavage dosing therapy for 6 days, and the mice are monitored daily for body weight. Mice were sacrificed on day 10 and DAI results were examined the day before sacrifice.

(2) Evaluation of colitis conditions: after sacrifice, the blind colon was dissected out and the colon length and appearance changes were recorded; taking intestinal tract tissue 0.5cm away from anus and fixing with 4% paraformaldehyde, dehydrating, embedding, slicing, performing HE staining and PAS staining, and observing colon pathological change and inflammatory infiltration degree.

(3) After colon tissue homogenization treatment, qRT-PCR and WB detection are carried out, 0.05g of tissue sample is taken, 300 mul of RIPA lysate is added, then the tissue sample is ground by a homogenizer, and protein samples are extracted to detect p-p65 and p-stat 3. 0.02g of tissue sample is taken, 1ml of Trizol RNA lysate is added, mRNA sample is extracted, and inflammatory factors IL-1 beta, IL-6 and TNF-alpha are detected.

(4) A set of t-tests was performed with GraphPad Prism 5.^*p<0.05, representing significant statistical significance;^**p<0.01,^***p<0.001,^****p<0.0001, indicates a high significance.

3. The experimental results are as follows:

(1) the effect of loganin on inflammatory indicators of DSS-induced acute enteritis in mice, including: body weight, DAI, and colon length. As shown in fig. 1 and 2, the DSS-induced acute enteritis model was successful, and loganin significantly reduced weight loss due to acute enteritis, and the effect was comparable to that of the positive control group SASP. In addition, loganin can reduce the disease activity index of DSS-induced acute enteritis (as shown in figure 3). At the same time, the therapeutic effect of loganin is also reflected in the change of colon length, as shown in fig. 4 and 5, which inhibits inflammatory pathological shortening of the colon (n-6,^**p<0.01,^****p<0.0001). The above indexes show that the treatment effect of the high-concentration group and the low-concentration group is not obviously different.

(2) Influence of loganin on the pathological indicators of colitis: the mucosa of the model group is damaged and has slight edema,a large number of inflammatory cells were infiltrated and the large intestine glands were lost and the arrangement of colonic epithelial cells was disturbed (as shown in fig. 6). Scoring based on the degree of change in mucosal structure, degree of inflammatory cell infiltration, degree of epithelial defect, and degree of goblet cell loss, the results are shown in fig. 7, with logarithmin-treated histopathological activity scoring lower than that of the model group (n-3,^**p<0.01,^***p<0.001). And further by PAS staining (as shown in fig. 8), the loganin stem cell population had significantly more goblet cells than the model population (as shown in fig. 9, n-3,^**p<0.01,^***p<0.001)。

(3) effect of loganin on the expression level of inflammatory factors of colon tissue. As shown in figures 10-12, RT-PCR detects the expression of three classical proinflammatory factors, IL-1 beta, IL-6 and TNF-alpha, and the result proves that loganin can effectively inhibit the expression of the inflammatory factors after treatment.

(4) The regulation and control of loganin on the protein expression of key proteins p-p65 and p-STAT3 of the inflammatory pathway NF- к B/STAT3 referring to FIGS. 13 and 14, WB test results demonstrate that loganin can inhibit the expression of p-p65 and p-STAT3 after intervention compared with model groups (see: FIGS.)^*p<0.05，^***p<0.001，^****p<0.001)。

Example 2 in vitro cell experiments

1. The main experimental materials: colon cancer cell line HCT116, purchased from Shanghai cell Bank, McCOY's 5A medium purchased from Sigma and Samorfei, and other reagents purchased from Shanghai assist in san Francisco Ltd and MCE in America.

2. Experimental methods

(1) And (3) detecting the activity of the cells: two colon cancer cells were cultured at 0.5 × 10⁴And each well is planted in a 96-well plate, after the iron wall of the cell, the drug adding treatment is started, the concentration gradient of loganin is 0, 0.5 mu m, 1 mu m, 5 mu m and 12.5 mu m, and after 24 hours, 20 mu l of MTT solution (5mg/ml in PBS) is added in each well. Incubation was continued for 4h, the culture was terminated, and the culture supernatant in the wells was carefully aspirated, after centrifugation was required for the suspension cells, and the culture supernatant in the wells was aspirated. Add 150. mu.l DMSO/well and shake for 10 minutes to fully melt the crystals. Selecting 490nm wavelength, and detecting by enzyme linked immunosorbent assayThe absorbance of each well was measured and the results were recorded.

(2) Extracting total RNA of cells and detecting Real time PCR: HCT116 colon cancer cell line at 5x10⁵And (2) inoculating the cells/well, after the cells are attached to the wall, replacing a serum-free culture medium, adding Logan in (12.5 mu m) for pretreatment for 16h, adding LPS (2 mu g/ml) after 16h for treatment for 3h, collecting samples, extracting a control group, an LPS group and total RNA of a medicine group by using a assist RNA rapid extraction kit, inverting, and detecting inflammatory factors IL-1 beta, IL-6, TNF-alpha and IFN-gamma by Real time PCR.

3. The experimental results are as follows:

(1) loganin intervenes in the regulation of colon cancer cell proliferation activity. As shown in FIG. 15, MTT assay found that loganin treatment had no significant effect on the proliferative activity of cells after 24h treatment with no concentrations (0, 0.5 μm, 1 μm, 5 μm and 12.5 μm) of loganin in colon cancer cells HCT116 as the study subjects.

(2) The effect of loganin intervention on inflammatory factor expression in the LPS-induced acute enteritis model. As shown in FIG. 16, in HCT116 and Caco-2 cells, loganin (12.5 μm) was added for pre-protection 16h in advance, LPS (2ug/ml) was added for two to three h before sampling, and finally the expression of the inflammatory factors IL-1 β, IL-6, TNF- α and IFN- γ was examined. RT-PCR results prove that after LPS induction, the expression of the inflammatory factors is increased, and meanwhile, the expression of the inflammatory factors can be obviously inhibited after loganin is added for treatment.

Claims

1. Application of loganin in preparing medicine for treating acute enteritis is provided.

2. The use of claim 1, wherein the acute enteritis is induced by dextran sulfate sodium salt.

3. The use of claim 1, wherein the acute enteritis is acute inflammation of colon cancer cells induced by lipopolysaccharide.