Application of 4-chloro-3-ethylphenol in the preparation of drugs for preventing or treating ulcerative colitis and sepsis

Abstract

The present invention discloses the use of 4-chloro-3-ethylphenol in the preparation of drugs for preventing or treating ulcerative colitis and sepsis, and relates to the field of biomedical technology. It is experimentally confirmed in the present invention that the ryanodine receptor (RyRs) agonist 4-chloro-3-ethylphenol has significant effects in preventing or treating ulcerative colitis and sepsis, and 4-chloro-3-ethylphenol is a clinically potential drug for the preparation of drugs for preventing or treating ulcerative colitis and sepsis; meanwhile, using 4-chloro-3-ethylphenol as a reliable tool can clearly distinguish the two components of store-operated calcium channels (SOCE), that is, inducing ER / Ca<supgt;2+< / supgt; release through RyR, but blocking the entry of Ca<supgt;2+< / supgt; through SOC in vascular endothelial cells, having a dual effect; further experiments confirm that pure endothelial cell RyR / ER / Ca<supgt;2+< / supgt> release is a unique pathway for regulating EDH-mediated arterial vasodilation in a healthy state and has an anti-inflammatory effect on ulcerative colitis and sepsis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically relates to the application of 4-chloro-3-ethylphenol in the preparation of drugs for preventing or treating ulcerative colitis and sepsis. Background Art

[0002] Ulcerative Colitis is an inflammatory bowel disease mainly characterized by inflammation and ulcerative lesions in the mucosa and submucosa of the rectum and sigmoid colon. The main manifestations are repeated severe abdominal pain, diarrhea, rectal bleeding and bloody stools. In severe cases, it can invade the entire colon and the terminal ileum. In recent years, the incidence and prevalence of ulcerative colitis have been increasing year by year and showing a trend of being younger. Due to its specific etiology still being unclear, coupled with its characteristics of long course, easy recurrence and many complications, there is still no good treatment strategy so far, and it has been listed as a modern intractable disease by the World Health Organization.

[0003] For the treatment of ulcerative colitis, the currently commonly used intervention drugs in clinical practice are mainly 5-aminosalicylic acid, steroid hormones and immunosuppressants. Although these three drugs have good short-term efficacy, they still have limitations such as poor long-term efficacy, high recurrence rate after drug withdrawal and large adverse reactions. Importantly, ulcerative colitis is prone to repeated development and aggravation, and there is even a risk of canceration if not intervened early. The long treatment cycle of colitis also brings a serious economic burden to the patient's family.

[0004] Sepsis is a systemic inflammatory response syndrome caused by infection and is one of the main causes of child death globally. In recent years, although significant progress has been made in anti-infection treatment, intensive care and organ function support technologies, the in-hospital mortality rate of sepsis patients is still relatively high, and the serious consequences of sepsis bring great psychological panic to patients and their families, which is not conducive to the further treatment of the disease.

[0005] Currently, relevant research shows that the occurrence and development of ulcerative colitis and sepsis are both related to Ca 2+ homeostasis disorder, but its pathogenesis is very complex and has not been fully elucidated. There is an urgent need to develop effective new drugs and clarify its pathogenesis; at the same time, no effective drugs for specifically treating the above two diseases have been successfully developed clinically so far, which is a severe challenge faced by the fields of biomedicine and critical care medicine.

[0006] Therefore, for ulcerative colitis and sepsis, how to develop effective new drugs is a technical problem that needs to be solved urgently by the current technicians in this field.

[0007] The information disclosed in this background section is only intended to enhance the overall understanding of the background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0008] In view of the above technical problems, embodiments of the present invention provide the use of 4-chloro-3-ethylphenol in the preparation of drugs for preventing or treating ulcerative colitis and sepsis to solve the problems raised in the above background section; wherein, 4-chloro-3-ethylphenol, abbreviated as 4-CEP, has a molecular formula of C8H9ClO, and its chemical structural formula is as Figure 1 shown.

[0009] Use of 4-chloro-3-ethylphenol in the preparation of drugs for preventing or treating ulcerative colitis.

[0010] Preferably, 4-chloro-3-ethylphenol is used to reduce the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the body.

[0011] Preferably, 4-chloro-3-ethylphenol is used to maintain the body weight of patients with ulcerative colitis; 4-chloro-3-ethylphenol is used to maintain the colon length of patients with ulcerative colitis; 4-chloro-3-ethylphenol is used to reduce the diarrhea and bloody stool conditions in patients with ulcerative colitis.

[0012] Use of 4-chloro-3-ethylphenol in the preparation of drugs for preventing or treating sepsis.

[0013] Preferably, 4-chloro-3-ethylphenol is used to improve the survival rate of patients with sepsis.

[0014] Preferably, 4-chloro-3-ethylphenol is used to relieve vasodilation disorders.

[0015] Preferably, 4-chloro-3-ethylphenol is used to reduce the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in patients with sepsis.

[0016] Preferably, the cause of sepsis is bacterial or inflammatory infection caused by one of the diseases such as pneumonia, cholangitis, peritonitis, urinary tract infection, abscess, and severe trauma.

[0017] Use of 4-chloro-3-ethylphenol in the preparation of drugs for preventing or treating target organ damage caused by sepsis, and 4-chloro-3-ethylphenol is used to prevent or treat liver and kidney function damage and relieve organ dysfunction.

[0018] Use of 4-chloro-3-ethylphenol in the preparation of RyR receptor agonist drugs.

[0019] The application of 4-chloro-3-ethylphenol provided by the embodiments of the present invention in the preparation of drugs for preventing or treating ulcerative colitis and sepsis has the following beneficial effects:

[0020] 1. It is confirmed by experiments of the present invention that 4-chloro-3-ethylphenol has a significant effect in preventing or treating ulcerative colitis and sepsis, and 4-chloro-3-ethylphenol is a clinically potential drug for preparing drugs for preventing or treating ulcerative colitis and sepsis;

[0021] 2. As a reliable tool, 4-chloro-3-ethylphenol can clearly distinguish two components of the store-operated calcium channel (SOCE), that is, induce ER / Ca 2+ release through RyR, but block the entry of Ca 2+ through SOC in vascular endothelial cells, having a dual effect;

[0022] 3. Further experiments illustrate that the pure endothelial cell RyR / ER / Ca 2+ release is a unique pathway regulating EDH-mediated arterial vasodilation in the healthy state, and this pathway has an anti-inflammatory effect on ulcerative colitis and sepsis. Description of the Drawings

[0023] Figure 1 is the chemical structural formula of 4-chloro-3-ethylphenol;

[0024] Figure 2 is the experimental result of the effect of 4-chloro-3-ethylphenol on the calcium signal of vascular endothelial cells HUVEC in vitro;

[0025] Among them, Figure 2 A is the result of the change in Ca 2+ signal induced by 4-chloro-3-ethylphenol (100 μM) under the condition of 0 Ca 2+ ; Figure 2 B is the result of the change in Ca 2+ signal induced by 4-chloro-3-ethylphenol (100 μM) after applying the selective inhibitor dantrolene (10 μM) of RyR1 / 3 under the condition of 0 Ca 2+ ; Figure 2 C is the difference statistical chart of Figures A and B;

[0026] Figure 3 is the experimental result of the effect of 4-chloro-3-ethylphenol on the secretion of inflammatory factors induced by LPS in vascular endothelial cells HUVEC;

[0027] Among them, Figure 3 A-C are the effects of applying 4-chloro-3-ethylphenol on the secretion of IL-1β, IL-6, and TNF-α inflammatory factors induced by LPS; Figure 3D-F show the effects of 4-chloro-3-ethylphenol on the secretion of inflammatory factors IL-1β, IL-6, and TNF-α after applying the selective inhibitor of RyR1 / 3, dantrolene, or the SOC channel inhibitor GSK7975A;

[0028] Figure 4 show the experimental results of the effect of 4-chloro-3-ethylphenol on ulcerative colitis in mice;

[0029] Among them, Figure 4 A shows the results of the body weight changes of mice in different groups; Figure 4 B shows the results of the fecal score of different groups; Figure 4 C shows the results of the changes in the colon length of mice in different groups;

[0030] Figure 5 show the experimental results of the preventive effect of 4-chloro-3-ethylphenol on sepsis in mice;

[0031] Figure 6 show the detection results of the microvascular dilation ability of mice when 4-chloro-3-ethylphenol is used to prevent sepsis in mice;

[0032] Among them, Figure 6 A shows the relaxation effect of ACh on the microvessels of sham-operated, model, and drug-pretreated CLP sepsis mice; Figure 6 B is Figure 6 A statistical chart of the maximum percentage of vascular dilation in different groups; Figure 6 C is Figure 6 A statistical chart of the area under the curve of the vascular dilation effect in different groups; Figure 6 D shows the relaxation effect of ACh on the microvessels of sham-operated, model, and drug-pretreated CLP sepsis mice in the presence of the NO and PGI2 inhibitors L-NNA and indomethacin; Figure 6 E is Figure 6 D statistical chart of the maximum percentage of vascular dilation in different groups; Figure 6 F is Figure 6 D statistical chart of the area under the curve of the vascular dilation effect in different groups;

[0033] Figure 7 show the detection results of the expression levels of inflammatory factors in mice when 4-chloro-3-ethylphenol is used to prevent sepsis in mice;

[0034] Among them, Figure 7 A shows the detection of the content of the secreted inflammatory factor TNF-α in each group of sera; Figure 7 B shows the detection of the content of the secreted inflammatory factor IL-6 in each group of sera; Figure 7 C shows the detection of the content of the secreted inflammatory factor IL-1β in each group of sera;

[0035] Figure 8 Experimental results of the effect of 4-chloro-3-ethylphenol on the survival time of CLP-induced septicemia mice;

[0036] Figure 9 Experimental results of the effect of 4-chloro-3-ethylphenol on the vasodilatory ability of CLP-induced septicemia mice;

[0037] Among them, Figure 9 A-C are the sham operation group, the model group, and the group administered the drug 3 hours after surgery. Statistical charts of the arteriolar microvascular dilation response, maximum dilation percentage, and area under the curve of ACh on CLP-induced septicemia mice; Figure 9 D-F are the sham operation group, the model group, and the group administered the drug 6 hours after surgery. Statistical charts of the arteriolar microvascular dilation response, maximum dilation percentage, and area under the curve of ACh on CLP-induced septicemia mice; Figure 9 G-I are the sham operation group, the model group, and the group administered the drug 12 hours after surgery. Statistical charts of the arteriolar microvascular dilation response, maximum dilation percentage, and area under the curve of ACh on CLP-induced septicemia mice;

[0038] Figure 10 Detection results of the expression levels of inflammatory factors in CLP-induced septicemia mice treated with 4-chloro-3-ethylphenol;

[0039] Among them, Figure 10 A is the detection of the content of serum inflammatory factor IL-1β in different groups; Figure 10 B is the detection of the content of serum inflammatory factor IL-6 in different groups; Figure 10 C is the detection of the content of serum inflammatory factor TNF-α in different groups;

[0040] Figure 11 Experimental results of the effect of 4-chloro-3-ethylphenol on the liver and kidney functions of CLP-induced septicemia mice;

[0041] Among them, Figure 11 A is alanine aminotransferase; Figure 11 B is aspartate aminotransferase; Figure 11 C is blood urea nitrogen; Figure 11 D is creatinine; Figure 11 E is diamine oxidase; Figure 11 F is lactic acid. Specific embodiments

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0043] In view of the above technical problems, the embodiments of the present invention provide the use of 4-chloro-3-ethylphenol in the preparation of drugs for preventing or treating ulcerative colitis and sepsis, so as to solve the problems raised in the above background art.

[0044] Example 1: Experiment on the effect of 4-chloro-3-ethylphenol on the calcium signal of vascular endothelial cells HUVEC in vitro

[0045] 1. Experimental materials:

[0046] 4-chloro-3-ethylphenol was purchased from MCE Company; dimethyl sulfoxide (DMSO) was purchased from SIGMA brand; calcium fluorescence detection reagent Fura-2, AM was purchased from Solarbio Company; Dantrolene was purchased from MCE Company.

[0047] 2. The experimental reagents were prepared as follows:

[0048] (1) Cell culture medium: By volume percentage, the medium contained 89% endothelial cell culture medium, 10% fetal bovine serum, and 1% streptomycin / penicillin;

[0049] (2) Phosphate buffer solution (PSS): Dissolve 0.818 g of NaCl, 0.037 g of KCl, 0.022 g of CaCl2, 0.238 g of HEPES, and 0.18 g of glucose in 90 mL of double-distilled water, adjust the pH to 7.4, and make up the volume to 100 mL for standby;

[0050] (3) Preparation of the calcium ion dye Fura-2, AM fluorescent dye stock solution (5 mM): 50 μg / vial, add 6 μL of DMSO and 4 μL of Pluronic F127 (20% in DMSO), and ultrasonically oscillate for 30 s to dissolve it completely; Preparation of the working solution (5 μM), take 1 μL of Fura-2, AM (5 mM) and dissolve it in 999 μL of PSS, mix well, and prepare it immediately before use.

[0051] 3. Experimental method:

[0052] (1) Culture HUVEC cells on the culture medium of a 15 mm diameter round transparent cell slide. When the cell density reaches about 80%, proceed to the next step;

[0053] (2) After washing the HUVEC cells twice with PSS, add the Fura-2, AM dye and incubate at 37 °C for 1 h;

[0054] (3) After the incubation, wash three times with PSS, and then incubate in PSS for 30 min;

[0055] (4) Image under a fluorescence microscope, select blue excitation light and green received light; image at an interval of 3 s; after loading, add 4-chloro-3-ethylphenol to make its final concentration reach 100 μM, image and record the green fluorescence intensity of the cells, and count the change rate of calcium fluorescence. The results are as Figure 2 shown.

[0056] Here it should be noted that dantrolene is a selective inhibitor of RyR and can inhibit the release of Ca 2+ from the endoplasmic reticulum (ER).

[0057] Fura-2, AM is a commonly used calcium fluorescence probe that can specifically bind to Ca 2+ (the binding ratio is 1:1) and emit fluorescence. The intensity of the emitted fluorescence has a quantitative relationship with the concentration of the bound Ca 2+ and is used to calculate the intracellular Ca 2+ concentration.

[0058] The ER is an important organelle in the cell, responsible for synthesizing, folding, and transporting proteins, and is also a Ca 2+ storage reservoir; the ER plays an important role in cell signal transduction through Ca 2+ reserves.

[0059] RyR (Ryanodine Receptor) is a type of calcium channel present on the endoplasmic reticulum membrane and is responsible for regulating the release of Ca 2+ from the ER; RyR1 and RyR3 are two different subtypes of receptors that play roles in different types of cells respectively.

[0060] SOC (Store-Operated Calcium Entry) is a calcium channel that is usually activated when the ER / Ca 2+ concentration decreases, allowing external Ca 2+ to enter the cell and restore the calcium reserve in the ER.

[0061] 3. Analyze the data:

[0062] Statistically analyze the relationship between the change rate of calcium fluorescence signal and the changes in time and concentration. The results are as Figure 2 shown in A. In the absence of extracellular Ca 2+ (0Ca 2+ ), 100 μM 4-chloro-3-ethylphenol induces the release of ER / Ca 2+ into the cytoplasm, but when the extracellular Ca 2+ is restored to 2 mM (2Ca 2+ ), due to the entry of Ca 2+ through SOC, it cannot induce a further increase in [Ca 2+ cyt .​

[0063] From Figure 2 Figures B and 2C, it can be seen that dantrolene (10 μM), a selective inhibitor of RyR1 / 3, can significantly attenuate 4-chloro-3-ethylphenol-induced Ca 2+ release, but Ca 2+ does not enter HUVEC cells, which strongly indicates that 4-chloro-3-ethylphenol-induced Ca in the ER 2+ is released through RyR3 rather than entering the cell through SOC, verifying the endothelial effect of 4-chloro-3-ethylphenol at the cellular level.

[0064] Example 2: Experiment on the effect of 4-chloro-3-ethylphenol on the secretion of inflammatory factors induced by LPS in vascular endothelial cells HUVEC

[0065] 1. Experimental materials:

[0066] LPS (Lipopolysaccharide) and GSK7975A were purchased from MCE; the Elisa kit for detecting inflammatory factors was purchased from R&D, USA.

[0067] 2. Experimental methods:

[0068] (1) Cultivate vascular endothelial cells HUVEC in a 12-well plate (density: 5×105 cells / well), and proceed to the next step after complete adherence;

[0069] (2) Divide into control group (Ctrl), model group (LPS, 1 μg / mL), LPS + 100 μM 4-chloro-3-ethylphenol, LPS + 100 μM 4-chloro-3-ethylphenol + 10 μM dantrolene, and LPS + 100 μM 4-chloro-3-ethylphenol + 10 μM GSK7975A groups, and culture for 24 h;

[0070] (3) Centrifuge at 1000 g for 20 min at 4°C, and take the supernatant;

[0071] (4) Detect the concentrations of TNF-α, IL-1β, and IL-6 in the supernatant according to the ELISA kit instructions and statistically analyze the levels of various inflammatory factors.

[0072] 3. Analyze data:

[0073] From Figure 3 it can be seen that compared with the control group, the levels of the three inflammatory factors in the model group were significantly increased, indicating that the HUVEC inflammation model was successfully constructed; compared with the model group, 4-chloro-3-ethylphenol significantly reduced the levels of inflammatory factors in the supernatant of the HUVEC cell inflammation model induced by LPS ( Figure 3(A-C), indicating its anti-inflammatory effect, but this effect can be significantly inhibited by the selective inhibitor dantrolene of RyR1 / 3, while there is no effect after using the SOC inhibitor GSK7975A ( Figure 3 D-F).

[0074] Example 3: Experiment on the effect of 4-chloro-3-ethylphenol on ulcerative colitis in mice

[0075] 1. Experimental materials:

[0076] Dextran sulfate sodium (DSS) was purchased from MPbio, USA; the preparation method of 2.5% DSS solution was: dissolve 2.5 g of DSS in 100 mL of deionized water.

[0077] 2. Experimental methods:

[0078] (1) 6-week-old C57BL / 6 mice were randomly divided into a control group, a model group, and an experimental group;

[0079] An ulcerative colitis model was induced by allowing C57BL / 6 mice to freely drink 2.5% DSS (mass percentage concentration) solution for 7 days. While freely drinking the DSS solution, each group of mice was treated with drugs at corresponding doses, that is, the control group mice were given free drinking water and diet, and at the same time, they were gavaged with normal saline; the model group mice were given a solution containing 2.5% DSS for free drinking and diet, and at the same time, they were gavaged with normal saline; the mice in experimental group 1 were given 2.5% DSS solution for free drinking, and at the same time, they were gavaged with 4-chloro-3-ethylphenol (5 mg / kg / day) 7 days before giving DSS; the mice in experimental group 2 were given 2.5% DSS solution for free drinking, and at the same time, they were gavaged with 4-chloro-3-ethylphenol (5 mg / kg / day) and dantrolene (5 mg / kg / day) 7 days before giving DSS; the mice in experimental group 3 were given 2.5% DSS solution for free drinking, and at the same time, they were gavaged with dantrolene (5 mg / kg / day) 7 days before giving DSS;

[0080] (2) At the end of the experiment, the mice were sacrificed by cervical dislocation, and the abdomen was opened to measure the colon length; the results were as Figure 4 shown. In the above statistical analysis chart, significant differences were indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns represented no statistical difference.

[0081] 3. Analyze the data:

[0082] The statistical results were as Figure 4 shown. From Figure 4It can be seen that the body weight of the mice in experimental group 1 was significantly higher than that of the mice in the model group, indicating that 4-chloro-3-ethylphenol can improve the body weight of mice with ulcerative colitis. From Figure 4 It can be seen that the fecal score of experimental group 1 was significantly lower than that of the model group, indicating that 4-chloro-3-ethylphenol can improve the fecal condition of mice with ulcerative colitis. From Figure 4 It can be seen that the colon length of the mice in experimental group 1 was longer than that of the mice in the model group and was close to that of the control group, indicating that 4-chloro-3-ethylphenol can restore the colon length of mice with ulcerative colitis; Through Figure 4 Comparing experimental groups 2 and 3, it was found that dantrolene could reverse the therapeutic effect of experimental group 1, while dantrolene itself had no effect, indicating that 4-chloro-3-ethylphenol may improve ulcerative colitis by stimulating the RyR3 / ER / Ca 2+ release pathway.

[0083] Example 4: Experiment on the preventive effect of 4-chloro-3-ethylphenol on murine sepsis

[0084] 1. Experimental animals:

[0085] Sixty C57BL / 6 mice weighing 21-25 g were randomly divided into a sham operation group, a CLP group, and a 5 mg / kg 4-chloro-3-ethylphenol + CLP group (pretreated with 4-chloro-3-ethylphenol at 5 mg / kg / d for five days), with 20 mice in each group.

[0086] 2. Experimental methods:

[0087] (1) The drug pretreatment group was given 4-chloro-3-ethylphenol by gavage every day at a dose of 0.5 mg / kg; the model group and the sham operation group were given an equal volume of normal saline by gavage once a day for five consecutive days;

[0088] (2) The CLP sepsis model was constructed for the model group and the drug pretreatment group, and the specific procedure was as follows:

[0089] a. The mice were fasted for 12 h before the experiment;

[0090] b. After anesthesia, the animals were fixed supine on the operating board, the abdominal surgical area was routinely disinfected and depilated, and a 2-cm-long incision was made on the abdominal wall with a scalpel under sterile conditions. After entering the abdomen through the incision, the cecum was separated at a distance of 1 / 3 from the ileocecal valve and ligated with a No. 3 silk thread;

[0091] c. A No. 18 injection needle was used to puncture the ligated end, and a little feces was squeezed out, trying to avoid damaging blood vessels. Then the peritoneum and skin were sutured intermittently with a No. 4 silk thread, and at the same time, 50 mL / kg body weight of normal saline was immediately injected subcutaneously for shock resistance; the sham operation group only opened the abdominal cavity, but did not perform ligation and puncture operations on the cecum.

[0092] (3) Observe the mortality of mice at 12h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h after surgery and draw the survival curve. The results are as Figure 5 shown, and significant differences are indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns represents no statistical difference.

[0093] 3. Analyze the data:

[0094] It can be seen from Figure 5 that compared with the model group, the survival rate of mice in the 4-chloro-3-ethylphenol pretreatment group was significantly increased. This indicates that 4-chloro-3-ethylphenol can prevent sepsis in mice and improve the survival rate of septic mice.

[0095] Example 5: Detection of the microvascular dilation ability of mice when 4-chloro-3-ethylphenol is used to prevent sepsis in mice

[0096] 1. Experimental materials:

[0097] Acetylcholine (ACh) in the example was purchased from MCE Company.

[0098] 2. Experimental animals:

[0099] The grouping, drug administration treatment, and CLP modeling steps of the mice were the same as in Example 4.

[0100] 3. Experimental method:

[0101] (1) After CLP modeling for 12h, measure the dilation ability of the mesenteric artery microvessels of mice:

[0102] a. Sacrifice the mice by cervical dislocation, place them on the operating table with their abdomens up, open the abdominal wall, and isolate the mesenteric artery;

[0103] b. Slowly insert the first silver wire into the artery under the microscope, and put the artery together with the wire into another petri dish for standby;

[0104] c. First fix both ends of the silver wire on one end of the detection clip using micro forceps and a small screwdriver; bring the two measurement clips closer, hold the second silver wire with fiber forceps, insert it parallel and closely against the first silver wire. After the second silver wire is completely inserted, bring the clips closer and fix the silver wire at the other end of the measurement clip, and slightly separate the clips;

[0105] d. After heating and ventilating for 20 min, add norepinephrine to constrict the blood vessels;

[0106] e. Add ACh / PSS in ascending order of concentration (the concentration gradient range of ACh in the solution is 10 -8 -10 - 3 mol / L), and detect the tension after vascular dilation.

[0107] (2) The statistical results are as shown in Figure 6 , and significant differences are indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns represents no statistical difference.

[0108] 4. Analyze the data:

[0109] As shown in Figure 6 A-C, most of the vasorelaxant neurotransmitters induced by ACh in the vagus nerve of CLP sepsis mice are damaged, and 4-chloro-3-ethylphenol significantly restores the damaged vasorelaxation. As shown in Figure 6 D-F, in the presence of the NO and PGI2 inhibitors L-NNA and indomethacin, 4-chloro-3-ethylphenol can also restore the damaged vasorelaxation. At the same time, by comparing the maximum percentage of vasodilation and the area under the curve of the blood vessels in the sham operation group, model group, and drug pretreatment group, it is seen that the vasodilatory ability of the drug pretreatment group is significantly improved compared with the model group. These data indicate that 4-chloro-3-ethylphenol prevents sepsis by rescuing the EDH-mediated vasorelaxation mechanism.

[0110] Example 6: Detection of the expression level of inflammatory factors in mice when 4-chloro-3-ethylphenol is used to prevent sepsis in mice

[0111] 1. Experimental animals:

[0112] The grouping, drug administration treatment, and CLP modeling steps of the mice are the same as in Example 4.

[0113] 2. Experimental methods:

[0114] (1) After CLP modeling for 12 h, the mice were sacrificed by cervical dislocation, blood was collected from the eyeballs, and serum was taken after coagulation;

[0115] (2) Referring to the instructions of the Elisa kit, the content of inflammatory factors in the serum of each group of mice was detected;

[0116] (3) The statistical results are as shown in Figure 7 , and significant differences are indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001 indicates that ns represents no statistical difference.

[0117] 3. Analyze the data:

[0118] From Figure 7 It can be seen that the contents of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the serum of the model group were significantly increased compared with those of the sham operation group; the increases in the contents of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the serum of the mice in the drug pretreatment group were less than those of the model group. It shows that 4-chloro-3-ethylphenol can reduce the contents of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the serum caused by sepsis, and 4-chloro-3-ethylphenol can significantly control the inflammatory response caused by sepsis and prevent sepsis.

[0119] Example 7: Experiment on the effect of 4-chloro-3-ethylphenol on the survival period of CLP-induced septic mice

[0120] 1. Experimental animals:

[0121] 100 C57BL / 6 mice weighing 21 - 25 g were randomly divided into a sham operation group, a model group, a group administered with the drug 3 h after surgery, a group administered with the drug 6 h after surgery, and a group administered with the drug 12 h after surgery, with 20 mice in each group.

[0122] 2. Experimental methods:

[0123] (1) The sepsis model was constructed by CLP for the model group, the group administered with the drug 3 h after surgery, the group administered with the drug 6 h after surgery, and the group administered with the drug 12 h after surgery. The model construction process was the same as that in Example 4. The sham operation group only opened the abdominal cavity, but did not perform ligation and puncture operations on the cecum;

[0124] (2) The groups administered with the drug 3 h after surgery, the group administered with the drug 6 h after surgery, and the group administered with the drug 12 h after surgery were intragastrically administered with 4-chloro-3-ethylphenol at 0.5 mg / kg at 3 h, 6 h, and 12 h after surgery, respectively;

[0125] (3) The death conditions of the mice were observed at 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after surgery. The survival curves were drawn using Graphpad software and statistically analyzed;

[0126] (4) The results are as Figure 8 shown. The significant differences were determined using * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 indicates that ns represents no statistical difference.

[0127] 3. Analyze the data:

[0128] From Figure 8 It can be seen that none of the mice in the sham operation group died within 7 days; the mice in the model group started to die 12 h after modeling, and the 7-day survival rate was less than 20%. Compared with the model group, the 7-day survival rates of the groups administered at 3 h and 6 h after surgery were significantly higher than that of the model group, indicating that 4-chloro-3-ethylphenol has a good therapeutic effect on murine sepsis.

[0129] Example 8: Experiment on the effect of 4-chloro-3-ethylphenol on the vasodilatory ability of CLP-induced septic mice

[0130] 1. Experimental animals:

[0131] The grouping, drug administration treatment and CLP modeling steps of the mice were the same as those in Example 7.

[0132] 2. Experimental methods:

[0133] (1) 24 h after CLP modeling, the vasodilatory ability of the mesenteric artery microvessels was measured. The specific experimental steps were the same as those in step 3(1)a-e of Example 5;

[0134] (2) The statistical results are as Figure 9 shown, and significant differences were indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns represented no statistical difference.

[0135] 3. Data analysis:

[0136] From Figure 9 A, 9D and 9G, it can be seen that the percentage of vasodilation in the model group was only about 20%, indicating that the vasodilatory ability of the model group was severely impaired; the percentage of vasodilation in the groups administered at 3 h ( Figure 9 B and C), 6 h ( Figure 9 E and F) and 12 h ( Figure 9 H and I) after surgery increased with the increase in ACh concentration. The vasodilatory abilities of the groups administered at 3 h, 6 h and 12 h after surgery were significantly improved compared with the model group. The above results indicate that administration of 4-chloro-3-ethylphenol within 12 h after modeling can effectively improve EDH-mediated vasorelaxation in septic mice and restore blood perfusion in multiple organs to treat murine sepsis.

[0137] Example 9: Experimental detection of the effect of 4-chloro-3-ethylphenol on the expression levels of inflammatory factors in CLP-induced septic mice

[0138] 1. Experimental animals:

[0139] The grouping, drug administration treatment, and CLP modeling steps of the mice were the same as those in Example 7.

[0140] 2. Experimental method:

[0141] (1) After 24 hours of CLP modeling, the mice were sacrificed by cervical dislocation. Blood was collected from the orbital socket, and serum was obtained after blood coagulation.

[0142] (2) Referring to the instructions of the Elisa kit, the content of inflammatory factors in the serum of mice in each group was detected.

[0143] (3) The statistical results were as Figure 10 shown in, and significant differences were indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns represented no statistical difference.

[0144] 3. Analyze the data:

[0145] It can be seen from Figure 10 that the contents of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the serum of the model group were significantly increased compared with those of the sham operation group. The increase in the contents of pro-inflammatory factors IL-1β and TNF-α in the serum of the mice in the drug administration group at 3 hours after surgery and the drug administration group at 6 hours after surgery was less than that of the model group; the increase in the content of pro-inflammatory factor IL-6 in the serum of the drug administration group at 3 hours after surgery was less than that of the model group. The results indicate that 4-chloro-3-ethylphenol can significantly reduce the contents of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the serum caused by sepsis. At the same time, administering 4-chloro-3-ethylphenol within 6 hours after modeling can significantly control the inflammatory response caused by sepsis and treat sepsis.

[0146] Example 10: Experiment on the effects of 4-chloro-3-ethylphenol on the liver and kidney functions of CLP-induced septic mice

[0147] 1. Experimental materials:

[0148] The serum creatinine detection kit was purchased from Leagene; the commercial kits for serum alanine aminotransferase, aspartate aminotransferase, urea nitrogen, and lactate were from Nanjing Jiancheng Bioengineering Institute, China; the commercial kit for serum diamine oxidase was from Solarbio.

[0149] 2. Experimental animals:

[0150] The grouping, drug administration treatment, and CLP modeling steps of the mice were the same as those in Example 7.

[0151] 3. Experimental method:

[0152] (1) After 24 hours of CLP modeling, the mice were sacrificed by cervical dislocation. Blood was collected from the orbital socket, and serum was obtained after coagulation.

[0153] (2) Referring to the kit instructions, the contents of alanine aminotransferase, aspartate aminotransferase, urea nitrogen, creatinine, diamine oxidase, and lactic acid in the serum of mice in each group were detected.

[0154] (3) The statistical results are as Figure 11 shown, and significant differences are indicated by * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns represents no statistical difference.

[0155] 4. Analyze the data:

[0156] It can be seen from Figure 11 that the contents of alanine aminotransferase, aspartate aminotransferase, urea nitrogen, creatinine, diamine oxidase, and lactic acid in the serum of the model group were significantly increased compared with the sham operation group. The contents of alanine aminotransferase, aspartate aminotransferase, urea nitrogen, creatinine, diamine oxidase, and lactic acid in the serum of the drug administration groups at 3 h and 6 h after surgery were significantly lower than those in the model group, indicating that drug administration 3 - 6 h after modeling could effectively inhibit the increase in the contents of alanine aminotransferase, aspartate aminotransferase, urea nitrogen, creatinine, diamine oxidase, and lactic acid in the serum; the contents of alanine aminotransferase, creatinine, diamine oxidase, and lactic acid in the serum of the drug administration group at 12 h after surgery were lower than those in the model group, indicating that drug administration 12 h after modeling could effectively inhibit the increase in the contents of alanine aminotransferase, creatinine, diamine oxidase, and lactic acid in the serum. In summary, administering 4 - chloro - 3 - ethylphenol within 12 h after modeling could alleviate the liver and kidney function damage of septic mice to varying degrees, prevent or treat multiple organ dysfunction caused by sepsis, and treat sepsis.

[0157] In summary, the present invention has experimentally confirmed that 4 - chloro - 3 - ethylphenol can be used as a reliable tool to clearly distinguish the two components of SOCE, namely ER / Ca 2+ release and Ca 2+ entry through SOC, and has a dual effect. It further explains that the pure endothelial cell RyR / ER / Ca 2+ release is a unique pathway for regulating EDH - mediated arterial vasodilation in a healthy state, and this pathway has an anti - inflammatory effect on ulcerative colitis and sepsis.

[0158] The present invention for the first time reveals the following points compared with the prior art: 1) In a healthy state, only endothelial cell ER / Ca 2+ release may induce arterial vasodilation purely mediated by EDH; 2) Endothelial cell RyR / ER / Ca 2+Release may have an anti-inflammatory effect on ulcerative colitis and sepsis; 3) RyR / ER / Ca 2+ / EDH-mediated vasodilation may be a "friendly" pathway for preventing / treating ulcerative colitis and sepsis, but Ca 2+ entering through SOC may be an "enemy" pathway that exacerbates sepsis.

[0159] By using animal models, the present invention first discloses the use of 4-chloro-3-ethylphenol in the preparation of drugs for preventing or treating ulcerative colitis and sepsis: 1) 4-chloro-3-ethylphenol effectively prevents, treats, and alleviates ulcerative colitis by regulating the calcium signal in vascular endothelial cells, reducing the level of inflammatory factors, and accelerating the repair after intestinal mucosal injury; 2) 4-chloro-3-ethylphenol can maintain the body weight of mice with ulcerative colitis, restore the length of their colon, and improve the conditions of diarrhea and bloody stools; 3) In a CLP-induced sepsis mouse model, it was confirmed that 4-chloro-3-ethylphenol can significantly reduce the mortality rate of sepsis mice, reduce pro-inflammatory factors, improve multiple organ dysfunction, prevent and treat sepsis by improving the vasodilation of damaged blood vessels through EDH, and significantly prevent or treat liver and kidney function injuries caused by sepsis.

[0160] We provide strong evidence to support that 4-chloro-3-ethylphenol has dual cellular targets, which may be superior to other single-target drugs, such as dantrolene that only inhibits ER / Ca 2+ release and GKS7975A that only blocks Ca 2+ entry. Therefore, as a safe diagnostic drug for malignant hyperthermia, 4-chloro-3-ethylphenol can be used as a potential new drug and reused as a new therapeutic drug for preventing / treating ulcerative colitis and sepsis through a new RyR3 / ER / Ca 2+ release pathway.

[0161] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of 4-chloro-3-ethylphenol in the preparation of a drug for preventing or treating ulcerative colitis.

2. Use of 4-chloro-3-ethylphenol according to claim 1 in the preparation of a medicament for preventing or treating ulcerative colitis, characterized in that, 4-chloro-3-ethylphenol is used to reduce the expression levels of inflammatory factors TNF-α, IL-1β and IL-6 in the body.

3. Use of 4-chloro-3-ethylphenol according to claim 1 in the preparation of a medicament for preventing or treating ulcerative colitis, characterized in that, 4-chloro-3-ethylphenol is used to maintain the body weight of patients with ulcerative colitis; 4-chloro-3-ethylphenol is used to maintain the colon length of patients with ulcerative colitis; 4-chloro-3-ethylphenol is used to reduce the diarrhea and blood in the stool conditions of patients with ulcerative colitis.

4. Use of 4-chloro-3-ethylphenol in the preparation of a drug for preventing or treating sepsis.

5. Use of 4-chloro-3-ethylphenol according to claim 4 in the preparation of a drug for preventing or treating sepsis, characterized in that, 4-chloro-3-ethylphenol is used to improve the survival rate of patients with sepsis.

6. Use of 4-chloro-3-ethylphenol according to claim 4 in the preparation of a drug for preventing or treating sepsis, characterized in that, 4-chloro-3-ethylphenol is used to relieve vasodilation disorders.

7. Use of 4-chloro-3-ethylphenol according to claim 4 in the preparation of a drug for preventing or treating sepsis, characterized in that, 4-chloro-3-ethylphenol is used to reduce the expression levels of inflammatory factors TNF-α, IL-1β and IL-6 in patients with sepsis.

8. Use of 4-chloro-3-ethylphenol according to claim 4 in the preparation of a drug for preventing or treating sepsis, characterized in that, The cause of sepsis is bacterial or inflammatory infection caused by one of the diseases such as pneumonia, cholangitis, peritonitis, urinary tract infection, abscess, and severe trauma.

9. Use of 4-chloro-3-ethylphenol in the preparation of a medicament for preventing or treating target organ damage caused by sepsis, characterized in that, 4-chloro-3-ethylphenol is used to prevent or treat liver and kidney function damage and relieve organ dysfunction.

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