CN111870598A

CN111870598A - Application of coptisine in preparation of medicine for treating ulcerative colitis

Info

Publication number: CN111870598A
Application number: CN202010692388.1A
Authority: CN
Inventors: 李彩兰; 鲁强
Original assignee: Zhuhai Campus Of Zunyi Medical University
Current assignee: Zhuhai Campus Of Zunyi Medical University
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2020-11-03

Abstract

The invention belongs to the field of medicines, and discloses an application of coptisine in preparation of a medicine for treating ulcerative colitis. The invention shows that the coptisine has good treatment effect on the ulcerative colitis, and provides a new application of the coptisine, namely the coptisine is used for preparing a medicament for treating the ulcerative colitis; coptisine is a natural monomer component in coptis and has no reported obvious side effect, so that the coptisine has the characteristics of low toxicity and high safety when being used for preparing the medicine for treating ulcerative colitis.

Description

Application of coptisine in preparation of medicine for treating ulcerative colitis

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of coptisine in preparation of a medicine for treating ulcerative colitis.

Background

Ulcerative Colitis (UC) is one of Inflammatory Bowel Diseases (IBD), a chronic nonspecific disease of the rectum and colon with an undefined etiology. The clinical manifestations of the disease are diarrhea, abdominal pain, mucopurulent bloody stool, tenesmus and the like, and the disease has the characteristics of easy relapse, difficult cure and easy canceration, and is listed as one of the modern refractory diseases by the World Health Organization (WHO). In traditional Chinese medicine, UC is classified into intestinal weakness, diarrhea and diarrhea, and large intestine damp-heat is the main pathogenesis of UC. The disease is common in western countries, and in recent years, the incidence rate of UC in China is in a remarkably rising trend. At present, a recognized treatment medicament and a method with definite curative effect are lacked, and clinically used medicaments for preventing and treating UC comprise an immunosuppressant, amino salicylic acid, a biological agent, glucocorticoid and the like, which have certain curative effect but have the problems of serious side effect, easy relapse, low tolerance of patients and the like. Therefore, the research of effective and safe new drugs for preventing and treating UC and the elucidation of the effector mechanism thereof have become hot spots of relevant clinical research.

The traditional Chinese medicine Coptis is recorded in Shen nong Ben Cao Jing, and is dried rhizome of goldthread root (Coptis chinsis Franch), coptidis deltoidea C.Y. Cheng et Hisao or Coptis teetaWall (Coptis teetaWall) of Ranunculaceae. Radix coptidis is a key medicine for treating damp-heat dysentery in traditional Chinese medicine, has the effects of clearing heat and removing toxicity, eliminating dampness and stopping dysentery, and is good at entering middle energizer and large intestine to treat diarrhea and dysentery. Modern pharmacological research proves that the coptis chinensis has the effects of resisting inflammation, resisting bacteria, protecting stomach and intestine, resisting UC and the like, and quaternary amine isoquinoline alkaloids such as berberine, palmatine, coptisine, epiberberine, jateorhizine and the like are main active ingredients of the coptis chinensis, but the berberine is generally considered to be a main efficacy source for treating the stomach and intestine diseases.

Current research only shows that Coptisine (COP) has multiple biological activities of antioxidation, anti-inflammation, antibiosis, lipid metabolism regulation and the like, but the effect of coptisine on the aspect of ulcerative colitis is not reported, and no precedent for using coptisine in preparing a medicament for treating ulcerative colitis exists.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of coptisine in preparing the medicine for treating ulcerative colitis, the coptisine can obviously improve the symptoms of ulcerative colitis animals, has the effects of repairing intestinal mucosa epithelial barriers and relieving intestinal inflammation, and has obvious effect of preventing and treating ulcerative colitis.

The application of coptisine in preparing a medicament for treating ulcerative colitis is disclosed, wherein the structural formula of the coptisine is shown as a formula (I):

the molecular formula of the coptisine is C₁₉H₁₄NO₄And the molecular weight is 320.32.

Experiments of the invention show that coptisine can obviously improve the symptoms of weight loss, diarrhea, bloody stool, colon shortening and the like of ulcerative colitis mice, reduce proinflammatory cytokines and increase the expression of anti-inflammatory cytokines, and protect the integrity of colon mucosa by inhibiting the apoptosis of the colon mucosa and increasing the expression of tight junction protein, thereby relieving the ulcerative colitis.

A medicine for treating ulcerative colitis comprises coptisine as active ingredient, and pharmaceutically acceptable adjuvants.

Preferably, the pharmaceutically acceptable auxiliary materials comprise at least one of a solvent, a filler, a lubricant, a disintegrating agent, a buffering agent, a cosolvent, an antioxidant, a bacteriostatic agent, an emulsifier, a binder or a suspending agent.

Preferably, the content of coptisine in the medicine is 0.5-25% by mass.

Preferably, the preparation form of the medicine is tablets, granules, capsules or suppositories.

Preferably, the administration mode of the medicament is oral or rectal administration.

The invention adopts gastric lavage Dextran Sodium Sulfate (DSS) solution to induce and construct an ulcerative colitis model, and by comparing the treatment effect of mesalazine (5-ASA, which is commonly used for treating ulcerative colitis) and Coptisine (COP) on ulcerative colitis, the invention shows that the coptisine has better treatment effect under lower dosage.

Experiments show that the coptisine can obviously improve the damage condition of the colon tissue of the mouse with the ulcerative colitis. In particular to effectively restore the crypt structure, reduce the necrosis of the colon mucous epithelium, improve the edema degree of the submucosa, reduce the infiltration of inflammatory cells and reduce the MPO (myeloperoxidase) activity. Coptisine can also significantly reduce the proinflammatory cytokines including TNF-alpha, IL-1 beta, IL-6, IL-17 and IFN-gamma in colon tissues of mice with ulcerative colitis and increase the content of anti-inflammatory cytokines such as IL-10 and TGF-beta.

Coptisine can also repair the damage condition of the intestinal mucosal epithelium barrier of mice with DSS-induced ulcerative colitis. Specifically, the expression level of the colon tissue tight junction protein (ZO-1, ZO-2, occludin and claudin-1) is increased, the protein and gene expression of the pro-apoptotic protein Bax and the gene levels of Caspase-3 and Cytochrome c are inhibited, the gene and protein expression of the anti-apoptotic protein Bcl-2 is increased, and the Bax/Bcl-2 ratio is effectively reduced, so that the DSS-induced damage of the intestinal mucosa barrier function is improved.

Coptisine also inhibits the activation of the NF- κ B signaling pathway. Specifically, the activation of NF-kB signal channel is inhibited by obviously up-regulating the expression of intracytoplasmic p65 protein, down-regulating the expression of intracellular p65 protein and reducing the ratio of p-I kB alpha/I kB alpha.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention shows that the coptisine has good treatment effect on the ulcerative colitis, and provides a new application of the coptisine, namely the coptisine is used for preparing a medicament for treating the ulcerative colitis;

(2) compared with mesalazine with obvious side effects, coptisine is a natural monomer component in coptis and has no reported obvious side effects, so that the coptisine has the characteristics of low toxicity and high safety when being used for preparing the medicine for treating ulcerative colitis.

Drawings

FIG. 1 shows the effect of coptisine on DSS-induced symptoms in mice with ulcerative colitis; (FIG. 1A) daily weight changes from day 0 to day 8; (FIG. 1B) DAI score; (FIG. 1C) macroscopic view of colon length in mice; (FIG. 1D) Bar graph of colon length differences in mice.

FIG. 2 shows the effect of coptisine on colon histopathological injury and MPO activity in DSS-induced ulcerative colitis mice; (FIG. 2A) histopathological changes in colon tissue in mice, IFI: inflammatory infiltration; (fig. 2B) histopathological scoring; (FIG. 2C) MPO activity.

FIG. 3 shows the effect of coptisine on the expression of pro-inflammatory and anti-inflammatory cytokines in mice with DSS-induced ulcerative colitis; (FIG. 3A) TNF- α; (FIG. 3B) IFN-. gamma.; (FIG. 3C) IL-1 β; (FIG. 3D) IL-6; (FIG. 3E) IL-17; (FIG. 3F) IL-10; (FIG. 3G) TGF-. beta.s.

FIG. 4 shows the effect of coptisine on intestinal mucosal barrier function in mice with DSS-induced ulcerative colitis; (FIG. 4A) Western blot plot of apoptosis-associated proteins; (FIG. 4B) Bax protein expression; (FIG. 4C) Bcl-2 protein expression, (FIG. 4D) Bax/Bcl-2 ratio; (FIG. 4E) Bax mRNA levels; (FIG. 4F) Bcl-2mRNA levels; (FIG. 4G) Caspase-3 mRNA levels; (FIG. 4H) Cytochrome c mRNA levels.

FIG. 5 shows the effect of coptisine on the expression of tight junction protein in mice with DSS-induced ulcerative colitis; (FIG. 5A) Western blot of the expression of the mouse colon tissue structure claudin; (FIG. 5B) ZO-1; (FIG. 5C) ZO-2; (fig. 5D) Occludin; (FIG. 5E) Claudin-1.

FIG. 6 shows the effect of coptisine on the NF-. kappa.B signaling pathway in DSS-induced ulcerative colitis mice; (FIG. 6A) Western blot plot of NF- κ B signal pathway-associated proteins; (FIG. 6B) NF-. kappa. B p65 (nucleus) (FIG. 6C) NF-. kappa. B p65 (parenchyma); (FIG. 6D) p-I.kappa.Balpha/I.kappa.Balpha.

In each of the above figures, # # indicates P <0.01 compared to the normal group; compared to the model group, denotes P <0.05, denotes P < 0.01.

Control: blank group, DSS: model set, 5-ASA: mesalamine (200mg/kg), COP: coptisine (25, 50 and 100 mg/kg).

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Experimental drugs: coptisine (COP) was purchased from WUDIHUAZHENHUANGSHENGZHI GmbH; mesalamine (5-ASA) was purchased from Losan Pharma GmbH pharmaceutical.

Experimental reagent: dextran sulfate sodium (DSS, MW: 36000-50000) was purchased from MP Biomedicals, USA; the H & E staining kit is purchased from Wuhan doctor De bioengineering GmbH; myeloperoxidase (MPO) kit purchased from Nanjing institute of bioengineering; mouse ELISA kit: TNF-alpha, IL-6, IL-1 beta, IL-10, IL-17, IFN-gamma and TGF-beta, available from Beijing Chenglin Biotechnology Ltd; antibodies ZO-1, ZO-2, claudin-1, occludin, p65, p-I.kappa.Balpha, Bax, Bcl, GAPDH and horseradish peroxidase conjugated secondary antibodies were purchased from Affinity Biosciences.

Experimental animals: SPF-grade male BALB/c mice (20-23g) 6-8 weeks, purchased from Guangdong province medical laboratory animal center. Mice were acclimatized for 7 days at 22 + -2 deg.C and 50 + -10% relative humidity in a 12 hour light and 12 hour dark environment during which they were fed standard mouse chow and allowed free access to water.

Experimental grouping and dose design: prior to the start of the experiment BALB/c mice were recorded weighed and randomized into 7 groups by weight: normal group (Control), model group (DSS), mesalamine group (5-ASA, 200mg/kg), coptisine low dose group (COP-L, 25mg/kg), coptisine medium dose group (COP-M, 50mg/kg), coptisine high dose group (COP-H, 100mg/kg), 10 mice per group.

Establishment of mouse model for ulcerative colitis: BALB/c mice freely drink 3% dextran sodium sulfate DSS aqueous solution for 7 days, and an ulcerative colitis model is induced. While the model is established, each group of mice is treated by the corresponding drug, wherein the normal group and the model group are treated by the corresponding solvent (0.5% CMC-Na aqueous solution) of the therapeutic drug. The administration was continued for 7 days. All mice were sacrificed on day 8 by cervical dislocation.

Example 1

A coptisine medicament (tablet) for treating ulcerative colitis is prepared by the following steps:

taking 50g of coptisine, adding 48g of lactose and 75.4g of starch, uniformly mixing, using 35g of 7% starch slurry as an adhesive, carrying out wet granulation, drying, adding 1.6g of magnesium stearate, uniformly mixing, and pressing into 1000 tablets containing 50mg of coptisine per tablet, wherein the net weight of each tablet is 0.21 g.

Is administered orally for treating ulcerative colitis. The symptoms are as follows: abdominal pain, diarrhea, hematochezia, weight loss, etc.

Example 2

A coptisine medicament (capsule) for treating ulcerative colitis is prepared by the following steps:

taking 50g of coptisine, adding 98g of lactose and 125.4g of starch, uniformly mixing, using 35g of 7% starch slurry as an adhesive, carrying out wet granulation, drying, adding 1.6g of magnesium stearate, uniformly mixing, filling into No. 1 capsules, and preparing into 1000 capsules, wherein each capsule contains 50mg of coptisine, and the net weight of each capsule is 0.31 g.

Example 3

A coptisine medicament (granule) for treating ulcerative colitis is prepared by the following steps:

weighing 30g coptisine, adding into appropriate amount of beta-cyclodextrin to obtain clathrate, adding appropriate amount of sucrose powder, microcrystalline cellulose and starch slurry, mixing, gradually adding ethanol and water, granulating, and packaging. The specification is that each bag contains 50mg of coptisine, and each bag has a net weight of 7 g.

It can be administered orally with boiled water for treating ulcerative colitis. The symptoms are as follows: abdominal pain, diarrhea, hematochezia, weight loss, etc.

Example 4

A coptisine preparation (suppository) for treating ulcerative colitis is prepared by the following steps:

taking 20g of coptisine, adding auxiliary materials such as cocoa butter and the like, and preparing into suppository containing 50mg of coptisine.

Rectal administration for treating ulcerative colitis. The symptoms are as follows: abdominal pain, diarrhea, hematochezia, weight loss, etc.

Example 5

Disease Activity Index (DAI) score

In the experimental process, the weight change, the activity condition, the stool character and the bloody stool condition of the mouse are observed and recorded every day. DAI scores were scored according to the following criteria:

body weight change score: unchanged, 0 point; 1-5 percent reduction and 1 minute reduction; the reduction is 5 to 10 percent and 2 minutes; the reduction is 10 to 20 percent and 3 minutes; the reduction is more than 20 percent and 4 minutes.

Stool shape scoring: normal, 0 point; the feces are soft and 1 minute; wetting and softening the excrement for 2 minutes; half loose stool, 3 minutes; dilute stool, 4 points.

Bloody stool condition score: bloodless stool, 0 point; positive stool blood test, 2 points; bloody stool, 4 points.

DAI ═ 3 (weight loss fraction + stool shape fraction + bloody stool fraction).

Measurement of colon length: after the mice were sacrificed by cervical dislocation, the colon was dissected and obtained, and then, the length of the colon was measured, data was recorded and pictures were taken. Approximately 1cm of colon was taken and placed in 4% paraformaldehyde for subsequent H & E (hematoxylin-eosin) staining experiments, and the remaining colon tissues were stored at-80 ℃.

The study finds that in the test period, mice in the blank control group ingest water normally, and the hair is clean and glossy; the movement is sensitive and active, and the body weight keeps a stable trend. The mouse in the model group has sparse, upright and dull hair, is normally laid and crouched, has stable weight in the first 4 days and basically keeps unchanged; by day 5, the body weight of the model group mice decreased significantly and in a state of sustained decrease.

As shown in fig. 1A, each administration treatment group of coptisine and the positive drug mesalazine can significantly improve the weight loss of mice induced by DSS, and the effect of coptisine on inhibiting weight loss is also significantly better than that of the positive drug mesalazine.

As shown in fig. 1B, the DAI score of the model mice increased from day 1 and was in a continuously increasing state, and the score was significantly increased compared to the blank control group, and the difference was statistically significant (P < 0.05). Compared with the model group, the coptisine administration treatment group can significantly reduce the DAI score in a dose-dependent manner (P <0.05), and the effect of the coptisine medium-dose group and the coptisine high-dose group is better than that of the mesalazine group.

As shown in fig. 1C and 1D, the colon length was significantly shortened (P <0.05) in the model group mice compared to the normal group; after the coptisine and mesalazine medicines are used for treatment, the condition is recovered, and the colon length of each group of mice is obviously increased compared with that of a model group (P is less than 0.05).

In conclusion, coptisine can obviously improve macroscopic symptoms of mice of a glucan sodium sulfate DSS-induced ulcerative colitis model and has an anti-ulcerative colitis effect.

Example 6

Colon histopathology observations and scoring

A sample of the colon of the mouse fixed in 4% paraformaldehyde solution of example 5 was taken, dehydrated, paraffin-embedded, sectioned (thickness of 5 μm), stained with hematoxylin and eosin, and the colon tissue was observed under a microscope for routine pathological changes and scored according to the histopathological state (histopathological score):

degree of inflammation: no inflammation, 0 point; superficial inflammation, 1 point; minor ulcer, 2 points; ulcer evident, score 3.

Extent of crypt damage: normal shape, 0 point; a small amount of crypts deformed by 1 minute; moderate horizontal crypt distortion, 2 points; high level of crypt deformation, score 3; obvious crypt deletion, score 4.

Area of inflammatory infiltration: no inflammatory cell infiltration, 0 point; 10% of the fields showed low levels of inflammatory cell infiltration, 1 point; 10-25% of the visual field shows moderate level of inflammatory cell infiltration, 2 points; high levels of inflammatory cell infiltration were observed in 25-50% of the fields, 3 points; inflammatory cell infiltration was evident, in 4 points.

Depth of intestinal wall inflammation: no inflammation, 0 point; inflammation was limited to the mucosal layer, 1 point; the inflammation gradually deepens into the submucosa for 2 minutes; inflammation deepens into the lamina propria, in score 3.

The total score was varied from 0 (normal) to 14 (severe enteritis).

Determination of MPO Activity

Taking out colon tissues stored at-80 ℃, and mixing the colon tissues in a weight-volume ratio of 1: 19 adding the relevant reagents in the MPO kit to prepare 5% tissue homogenate, and calculating the MPO activity strictly according to the kit instruction.

As shown in fig. 2A, compared to the blank control group, the mesalamine group, and the coptisine administration treatment group, the mucosal structure of the model group mice was severely damaged, and the characteristics thereof included severe epithelial injury, decreased goblet cells, substantial disappearance of crypts or intestinal glands, severe edema of submucosa or muscular layer, and significant infiltration of inflammatory cells of submucosa; as shown in fig. 2B-C, higher histopathological score and MPO activity in the model group mice compared to the blank control group; in comparison to the model group (DSS), the coptisine-administered treatment group (25mg/kg, 50mg/kg and 100mg/kg) significantly restored crypt structure and reduced colonic mucosal epithelial necrosis in a dose-dependent manner, improved submucosal edema, reduced inflammatory cell infiltration, reduced histopathological score and reduced MPO activity (P < 0.05); compared with the mesalazine group (5-ASA, 200mg/kg), the coptisine high-dose group (COP-H, 100mg/kg) has less drug dosage, but better effects of reducing histopathological score and MPO activity, and the coptisine is proved to have better curative effect on treating ulcerative colitis.

Example 7

ELASA assay for inflammatory factors colonic tissue stored at-80 ℃ was removed and mixed at a weight to volume ratio of 1: 9 adding precooled PBS solution, homogenizing, 3500g, centrifuging for 15min at 4 ℃, taking supernatant, and respectively measuring the contents of inflammatory factors TNF-alpha, IL-6, IL-1 beta, IL-10, IL-17, IFN-gamma and TGF-beta strictly according to the operation of ELASA kit instructions.

As shown in FIG. 3, compared to the blank control group, the colon tissues of mice in the DSS-induced colitis model group had a significant increase (P <0.05) in proinflammatory cytokines (TNF-. alpha., IL-1. beta., IL-6, IL-17, and IFN-. gamma.) and a significant decrease (P <0.05) in anti-inflammatory cytokines (IL-10 and TGF-. beta.). Compared with the model group, the coptisine administration treatment group significantly reduced the 5 anti-inflammatory cytokines and increased the levels of the 2 anti-inflammatory cytokines in a dose-dependent manner (P <0.05), indicating that coptisine can improve UC symptoms by effectively inhibiting DSS-induced UC mouse inflammatory protein levels.

Example 8

RT-PCR detection of expression of related genes

Total RNA extraction: 100mg of mouse colon tissue was weighed into a 2mL EP tube, and 1mL Trizol (phenol as a main ingredient) was added. Shearing colon tissue with scissors, homogenizing, adding chloroform 200 μ L, covering the centrifugal tube, vortex mixing for 1min until the solution is milky white, and standing at room temperature for 10 min. Centrifuged at 15000g for 15min at 4 ℃. The centrifuge tube was carefully removed from the centrifuge, and the homogenate was divided into three layers at this time, i.e.: a colorless supernatant (containing RNA), an intermediate white protein layer (mostly DNA), and a colored lower organic phase. The supernatant was pipetted into another new 1.5mL EP tube. Adding 500 mu L of isopropanol into the supernatant, inverting the centrifuge tube upside down, and standing for 1 hour at-20 ℃. 12000g, 4 ℃ centrifugation for 5min, carefully discard the supernatant. Add 500. mu.L of absolute ethanol, wash the tube wall of the centrifuge tube gently upside down, centrifuge at 7500g for 5min at 4 ℃ and carefully suck the residual liquid with filter paper. Volatilizing ethanol at room temperature, adding 20 μ L DEPC water to dissolve RNA, and detecting RNA purity of the solution in a NanoDrop2000 ultramicro spectrophotometer with absorbance ratio OD260/OD280 controlled within 1.8-2.2.

Reverse transcription: taking the eight-row calandria with forceps, sucking 4 μ L of 4 XgDNA wister Mix solution to the bottom of the calandria with pipette gun, sequentially adding 4 μ L of the above RNA solution and 8 μ L of RNase free ddH₂Centrifuging the O solution, reacting for 2min at 42 ℃ in a PCR instrument, taking out, adding 4 mu L of 5 XHiScript IIqRT Supermix IIa solution, and reacting for 10min at 25 ℃ in the PCR instrument; at 50 ℃ for 30 min; 5min at 85 ℃; 4 ℃ and finally 40. mu.L of RNase free ddH per sample₂O, the synthesized cDNA was stored at-20 ℃.

Fluorescent quantitative PCR: after designing primers for mouse RT-PCR detection using NCBI on-line tool Primer-BLAST, primers were synthesized and purified by Compton Biotech (Shanghai) Ltd, the Primer sequences are shown in Table 1:

TABLE 1

Preparing a primer: and centrifuging each synthetic primer dry powder at 4 ℃ for 1min, adding corresponding amount of DEPC water according to the specification requirement, uniformly mixing by vortex, centrifuging to prepare 100 mu M mother solution, and diluting with DEPC water to obtain 10 mu M working concentration primer solution.

According to the forward primer: reverse primer: DEPC water ═ 1: 1: 3 to obtain a primer mixture

Taking eight rows of tubes with tweezers, adding 10 mu L of 2 XChamQ SYBR qPCR Master Mixa at the bottom of each tube, sequentially adding 5 mu L of primer mixture and 5 mu L of cDNA, centrifuging for 30s, and immediately carrying out PCR reaction according to the following conditions.

The specific reaction procedure is as follows: pre-denaturation, reaction at 95 ℃ for 30s, 1 cycle; PCR reaction, reaction at 95 ℃ for 10s, reaction at 60 ℃ for 30s, and 40 cycles; dissolution curve, reaction at 95 ℃ for 15s, reaction at 65 ℃ for 10s, and reaction at 95 ℃ for 30 s.

GAPDH is used as an internal reference gene, each reaction is repeated three times, and the cycle number Ct value is used for relative quantification. Fold of gene expression 2^-△△Ct，△△Ct＝(Ct Sample–Ct GAPDH)–(Ct Control–Ct GAPDH)。

Effect of coptisine on apoptosis in colon tissue of UC mice caused by DSS

As shown in FIGS. 4E-H, the expression levels of mRNA for Bax, Bcl-2, Caspase-3 (Caspase-3) and cytochrome C (cytochrome c) in colon tissues of each group of mice were respectively shown. Compared with a blank control group, the gene expression of the model group mice Bax, caspase-3 and cytochrome C is remarkably increased, and the gene expression of the model group mice Bcl-2 is remarkably reduced (P < 0.05). However, the coptisine-administered treatment groups (25mg/kg, 50mg/kg and 100mg/kg) significantly down-regulated Bax, Caspase-3, Cytochrome c and up-regulated Bcl-2 gene expression (P <0.05) in a dose-dependent manner. The results show that the coptisine can achieve the effect of resisting ulcerative colitis by reducing the apoptosis of intestinal mucosa cells.

Example 9

Western blot analysis

Extraction of total tissue protein: colonic tissue stored at-80 ℃ was removed and treated at a temperature of 100 mg: lysate (RIPA lysate: Cocktail: PMSF: phosphatase inhibitor a: phosphatase inhibitor B ═ 100: 2: 1: 1: 1) was added at a rate of 1mL, homogenized on ice, incubated on ice for 20min (with occasional vortexing), 14000g, centrifuged at 4 ℃ for 10min, and the supernatant was taken out into a 1.5mL EP tube and assayed for protein concentration using BCA kit.

Extraction of proteins in cytoplasm and nucleus: colon tissues stored at-80 ℃ were removed, total protein was extracted according to the instructions of the kit for extraction of nuclear and cytoplasmic proteins, and protein concentration was determined using the BCA kit.

Mixing the protein supernatant extracted in the following steps of 1: 4 adding 5 × loading buffer, denaturing at 100 deg.C for 5min, subpackaging and storing at-80 deg.C.

Preparing 8-10% polyacrylamide gel (SDS-PAGE) and 5% concentrated gel, and filling electrophoresis buffer after the concentrated gel is completely polymerized. Loading equal amount of sample by a microsyringe, adding 5 μ L marker on two sides of a lane to indicate the molecular weight of protein, performing 80V constant voltage electrophoresis for 30min, adjusting the voltage to 120V constant voltage electrophoresis, stopping electrophoresis when bromophenol blue line is run to the edge of gel, cutting off the required part of the gel, and transferring the gel onto a PVDF membrane by a wet method. The membrane was blocked with 5% skimmed milk powder at room temperature for 1 hour, and the PVDF membrane was immersed in TBST and washed 3 times for 10min each. The PVDF membrane was removed and incubated with the following antibodies at 4 ℃ overnight: NF-. kappa.Bp-p 65 (1: 1000), p65 (1: 1000), p-. kappa.B α (1: 1000), I-. kappa.B α (1: 1000), Bax (1: 1000), Bcl-2 (1: 1000), claudin-1 (1: 500), occludin (1: 500), ZO-1 (1: 500), ZO-2 (1: 500), GAPDH (1: 1000) and Histone H3 (1: 1000), and the protein bands were visualized using ECL chemiluminescence liquid after incubation with horseradish-labeled secondary antibody (1: 2000) at room temperature for 2 hours, the objective bands were analyzed using Image J software, and the relative expression amounts of the objective proteins were calculated using the GAPDH internal reference grayscale values.

The results of the experiment are expressed as mean ± standard deviation (mean ± SD). Statistical Analysis was performed using SPSS17.0 software, and comparisons of differences between groups were performed using One-Way Analysis of variance (One-Way Analysis of ANOVA), LSD for homogeneous differences between groups, and Dunnett's T3 for inhomogeneous differences.

Effect of coptisine on intestinal barrier function in colon tissue of UC mice caused by DSS

FIG. 4A is a western blot of the relevant apoptotic proteins, the expression of which was normalized by GAPDH; as shown in FIGS. 4B-D, each dose group of coptisine can significantly reduce Bax, up-regulate Bcl-2 protein expression, and significantly reduce the Bax/Bcl-2 ratio (P <0.05), and coptisine shows an obvious dose-dependent relationship, which indicates that coptisine can achieve the effect of resisting ulcerative colitis by reducing apoptosis of intestinal mucosa cells.

FIG. 5A shows a western blot of the expression of the tight junction protein of the mouse colon tissue structure, normalized to GAPDH. As shown in FIGS. 5B-E, the expression levels of Claudin (ZO-1, ZO-2, occludin and claudin-1) were significantly reduced in the DSS-treated model group mice as compared to the blank control group (P <0.05), indicating that the intestinal mucosal barrier structure was disrupted. Compared with the model group, the mesalamine group and coptisine administration treatment groups (25mg/kg, 50mg/kg and 100mg/kg) significantly increased the content of tight junction protein in a dose-dependent manner, and the content of the mouse tight junction protein in the coptisine high-dose group (COP-H, 100mg/kg) was similar to that in the blank control group, confirming that coptisine had a good effect of protecting the intestinal barrier function of colon tissues.

Effect of coptisine on NF-kB signal path of mice UC caused by DSS

FIG. 6A is a western blot of NF-. kappa.B signaling pathway proteins normalized to GAPDH for expression of the relevant proteins. As shown in FIGS. 6B-D, the expression levels of NF-. kappa. B p65 (in nucleus), NF-. kappa. B p65 (cytoplasm), and p-. kappa.B.alpha./I.kappa.B.alpha.proteins were determined. Compared with the blank control group, the ratio of P-I kappa B alpha/I kappa B alpha of mice in the DSS-induced colitis model group and the protein expression of P65 in nuclei are remarkably increased (P <0.05), the protein of cytoplasm P65 is remarkably reduced (P <0.05), and the fact that the phosphorylation of I kappa B alpha can be remarkably increased after DSS-induced ulcerative colitis and the P65 protein in the cytoplasm enters the nuclei to cause inflammation is shown. Compared with the model group, the mesalamine group (5-ASA, 200mg/kg) and the coptisine administration treatment group (25mg/kg, 50mg/kg and 100mg/kg) can obviously up-regulate the intracytoplasmic P65 protein expression, down-regulate the intracellular P65 protein expression and obviously reduce the P-I kappa B alpha/I kappa B alpha ratio (P <0.05), and the coptisine presents obvious dose dependence relationship. The research result indicates that coptisine can inhibit the activation of NF-kB signal channel by inhibiting DSS-induced phosphorylation of I kB alpha in colon tissues of UC mice and p65 protein in cytoplasm entering cell nucleus, thereby showing obvious UC-resistant curative effect.

Claims

1. The application of coptisine in preparing a medicine for treating ulcerative colitis is characterized in that the structural formula of the coptisine is shown as a formula (I):

2. the medicine for treating ulcerative colitis is characterized by taking coptisine as an active ingredient and further comprising pharmaceutically acceptable auxiliary materials.

3. The medicament of claim 2, wherein the pharmaceutically acceptable excipient comprises at least one of a solvent, a filler, a lubricant, a disintegrant, a buffer, a co-solvent, an antioxidant, a bacteriostatic agent, an emulsifier, a binder, or a suspending agent.

4. The medicament according to claim 2, wherein the coptisine content in the medicament is 0.5-25% by mass.

5. The medicament of claim 2, wherein the medicament is formulated in the form of tablets, granules, capsules or suppositories.

6. The medicament of claim 2, wherein the medicament is administered orally or rectally.