Application of 4-chloro-3-ethyl phenol in preparation of medicine for preventing or treating ulcerative colitis and septicopyemia

Abstract

The invention discloses application of 4-chloro-3-ethyl phenol in preparation of a medicine for preventing or treating ulcerative colitis and septicopyemia, and relates to the technical field of biological medicine. Experiments prove that the raney receptor (RyRs) agonist 4-chloro-3-ethyl phenol has a remarkable effect on preventing or treating ulcerative colitis and septicopyemia, and the 4-chloro-3-ethyl phenol is a clinical potential medicine for preventing or treating ulcerative colitis and septicopyemia; meanwhile, 4-chloro-3-ethylphenol is used as a reliable tool, two components of a calcium channel (SOCE) controlled by a calcium pool can be clearly distinguished, that is, ER / Ca < 2 + > release is induced through RyR, but Ca < 2 + > passing through SOC in vascular endothelial cells is blocked from entering, and dual effects are achieved; further experiments prove that pure endothelial cell RyR / ER / Ca < 2 + > release is a unique way for regulating EDH-mediated artery vasodilation in a healthy state, and has an anti-inflammatory effect on ulcerative colitis and septicopyemia.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of 4-chloro-3-ethylphenol in preparing medicines for preventing or treating ulcerative colitis and sepsis. Background Art

[0002] Ulcerative colitis is an inflammatory bowel disease characterized by inflammation and ulcerative lesions of the rectal and sigmoid colon mucosa and submucosa, and is mainly manifested by repeated severe abdominal pain, diarrhea, rectal bleeding and blood in the stool. In severe cases, it can invade the entire colon and terminal ileum. In recent years, the incidence and prevalence of ulcerative colitis have increased year by year and tend to be younger. As its specific cause is still unclear, and its long course, easy recurrence and many complications are characteristics, there is still no good treatment, and it has been listed as a modern intractable disease by the World Health Organization.

[0003] For the treatment of ulcerative colitis, the three most commonly used intervention drugs in clinical practice are 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 discontinuation of medication, and large adverse reactions. Importantly, ulcerative colitis is prone to repeated development and aggravation, and there is even a risk of cancer if it is 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 leading causes of death in children worldwide. In recent years, despite significant progress in anti-infection treatment, intensive care and organ function support technology, the in-hospital mortality rate of patients with sepsis is still high, and the serious consequences of sepsis have brought great psychological panic to patients and their families, which is not conducive to further treatment of the disease.

[0005] At present, relevant studies have shown that the occurrence and development of ulcerative colitis and sepsis are related to Ca 2+ However, their pathogenesis is very complex and has not yet been fully elucidated. There is an urgent need to develop effective new therapeutic drugs to clarify their pathogenesis. At the same time, no effective drugs have been successfully developed to treat the above two diseases, which is a severe challenge facing the fields of biomedicine and critical care medicine.

[0006] Therefore, how to develop effective new therapeutic drugs for ulcerative colitis and sepsis is a technical problem that technicians in this field urgently need to solve.

[0007] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0008] In view of the above technical problems, the present invention provides an application of 4-chloro-3-ethylphenol in the preparation of a drug for preventing or treating ulcerative colitis and sepsis, so as to solve the problems raised in the above background technology; wherein 4-chloro-3-ethylphenol, referred to as 4-CEP, has a molecular formula of C 8 H 9 ClO, chemical structure is as follows Figure 1 shown.

[0009] Application of 4-chloro-3-ethylphenol in preparing medicine 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 diarrhea and blood in stool in patients with ulcerative colitis.

[0012] Application of 4-chloro-3-ethylphenol in preparing medicine 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 a bacterial or inflammatory infection caused by one of pneumonia, cholangitis, peritonitis, urinary tract infection, abscess, severe trauma.

[0017] The invention discloses an application of 4-chloro-3-ethylphenol in the preparation of a drug for preventing or treating target organ damage caused by sepsis. 4-chloro-3-ethylphenol is used for preventing or treating liver and kidney damage and alleviating organ dysfunction.

[0018] Application of 4-chloro-3-ethylphenol in preparing RyR receptor agonist drugs.

[0019] The use of 4-chloro-3-ethylphenol provided in the embodiment of the present invention in the preparation of a drug for preventing or treating ulcerative colitis and sepsis has the following beneficial effects:

[0020] 1. The present invention has experimentally confirmed that 4-chloro-3-ethylphenol has a significant effect in preventing or treating ulcerative colitis and sepsis, and 4-chloro-3-ethylphenol is a potential clinical drug for preparing the prevention or treatment of ulcerative colitis and sepsis;

[0021] 2.4-Chloro-3-ethylphenol serves as a reliable tool to clearly distinguish the two components of store-operated calcium channels (SOCE), namely, ER / Ca induced by RyR. 2+ Release, but block Ca2+ transport through SOC in endothelial cells 2+ Entering, it has a dual role;

[0022] 3. Further experiments demonstrated that pure endothelial cell RyR / ER / Ca 2+ Release is a unique pathway that regulates EDH-mediated arterial vasodilation in health and has anti-inflammatory effects in ulcerative colitis and sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0025] in, Figure 2 A is 0Ca 2+ Under the condition, 4-chloro-3-ethylphenol (100 μM) induced Ca 2+ Signal change results; Figure 2 B is 0Ca 2+ Under these conditions, dantrolene (10 μM), a selective inhibitor of RyR1 / 3, was applied, and 4-chloro-3-ethylphenol (100 μM) induced Ca 2+ Signal change results; Figure 2 C is the statistical diagram of the difference between Figure A and B;

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

[0027] in, Figure 3 AC is the effect of 4-chloro-3-ethylphenol on the secretion of IL-1β, IL-6, and TNF-α inflammatory factors induced by LPS; Figure 3 DF is the effect of 4-chloro-3-ethylphenol on the secretion of inflammatory factors IL-1β, IL-6, and TNF-α after the application of dantrolene, a selective inhibitor of RyR1 / 3, or GSK7975A, an inhibitor of SOC channel;

[0028] Figure 4 These are the results of an experiment on the effect of 4-chloro-3-ethylphenol on ulcerative colitis in mice;

[0029] in, Figure 4 A shows the weight changes of mice in different groups; Figure 4 B is the stool scoring results of different groups; Figure 4 C shows the changes in colon length of mice in different groups;

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

[0031] Figure 6 The results are the test results of the microvascular dilation ability of mice when 4-chloro-3-ethylphenol was used to prevent sepsis in mice;

[0032] in, Figure 6 A is the relaxation effect of ACh on microvessels of CLP sepsis mice in sham operation group, model group and drug pretreatment group; Figure 6 B is Figure 6 A Statistical graph of the maximum vascular dilation percentage in different groups; Figure 6 C is Figure 6 A Statistical graph of the area under the vasodilation curve of different groups; Figure 6 D is between NO and PGI 2 The relaxant effect of ACh on microvessels of CLP septic mice in the sham operation group, model group and drug pretreatment group in the presence of inhibitors L-NNA and indomethacin; Figure 6 E is Figure 6 D Statistical graph of the maximum vascular dilation percentage in different groups; Figure 6 F is Figure 6 D. Statistical graph of the area under the vasodilation curve of different groups;

[0033] Figure 7 These are the test results of the expression levels of inflammatory factors in mice when 4-chloro-3-ethylphenol was used to prevent sepsis in mice;

[0034] in, Figure 7 A is the detection of the secretion content of serum inflammatory factor TNF-α in each group; Figure 7 B is the detection of the secretion content of serum inflammatory factor IL-6 in each group; Figure 7 C is the detection of the secretion content of serum inflammatory factor IL-1β in each group;

[0035] Figure 8 The results of the experiment on the effect of 4-chloro-3-ethylphenol on the survival of mice with sepsis induced by CLP;

[0036] Fig. 9 The results are the experimental results of the effect of 4-chloro-3-ethylphenol on the vasodilation ability of CLP-induced septic mice;

[0037] in, Fig. 9 AC is the statistical graph of the sham operation group, model group and the group given 3 hours after surgery, showing the dilation response of ACh to the arteriolar microvasculature of septic mice induced by CLP, the maximum dilation percentage and the area under the curve; Fig. 9 DF is the statistical graph of the sham operation group, model group and the group given 6 hours after surgery, the dilation response of ACh to the arteriolar microvasculature of septic mice induced by CLP, the maximum dilation percentage and the area under the curve; Fig. 9 GI is the statistical graph of the sham operation group, model group and 12-hour postoperative drug administration group, the microvascular dilation response of ACh to CLP-induced septic mice arterioles, the maximum dilation percentage and the area under the curve;

[0038] Fig.10 The results are the detection results of 4-chloro-3-ethylphenol on the expression levels of inflammatory factors in CLP-induced septic mice;

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

[0040] Fig.11 The experimental results of the effect of 4-chloro-3-ethylphenol on liver and kidney function in CLP-induced septic mice;

[0041] in, Fig.11 A is alanine aminotransferase; Fig.11 B is aspartate aminotransferase; Fig.11 C is urea nitrogen; Fig.11 D is creatinine; Fig.11 E is diamine oxidase; Fig.11 F is lactic acid. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within 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 a drug for preventing or treating ulcerative colitis and sepsis, so as to solve the problems raised in the above background technology.

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

[0045] 1. Experimental materials:

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

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

[0048] (1) Cell culture medium: The culture medium contains 89% endothelial cell culture medium, 10% fetal bovine serum, and 1% streptomycin / penicillin by volume;

[0049] (2) Phosphate buffer (PSS): 0.818 g NaCl, 0.037 g KCl, 0.022 g CaCl 2 , 0.238g HEPES, 0.18g glucose, dissolved in 90mL double distilled water, adjusted pH to 7.4, fixed to 100mL, set aside;

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

[0051] 3. Experimental methods:

[0052] (1) HUVEC cells were cultured on a 15 mm diameter round transparent cell slide and the cell density reached about 80% before proceeding to the next step;

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

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

[0055] (4) Imaging was performed under a fluorescence microscope, with blue excitation light and green receiving light selected; the imaging interval was 3 s; 4-chloro-3-ethylphenol was added to make the final concentration reach 100 μM, the green fluorescence intensity of the cells was recorded by imaging, and the change rate of calcium fluorescence was calculated. The results are as follows: Figure 2 shown.

[0056] It should be noted here that dantrolene is a selective inhibitor of RyR, which can inhibit Ca 2+ release.

[0057] Fura-2, AM is a commonly used calcium fluorescent probe that can specifically bind to Ca 2+ (The binding ratio is 1:1) and emits fluorescence, the intensity of which is related to the binding of Ca 2+ The concentration of Ca is quantitatively related to that of 2+ concentration.

[0058] ER is an important organelle in cells that is responsible for the synthesis, folding and transport of proteins and is also a Ca 2+ Storage depot; ER via Ca 2+ Reserves play an important role in cell signaling.

[0059] RyR (Ryanodine Receptor) is a type of calcium channel present on the endoplasmic reticulum membrane, responsible for regulating Ca 2+ Release; RyR1 and RyR3 are two different subtypes of receptors, each playing a role in different types of cells.

[0060] SOC (Store-Operated Calcium Entry) is a calcium channel that is usually located in the ER / Ca 2+ It is activated when the concentration decreases, allowing external Ca 2+ Enters the cell and restores calcium stores in the ER.

[0061] 3. Analyze the data:

[0062] The relationship between the change rate of calcium fluorescence signal and time and concentration was statistically analyzed. The results are as follows Figure 2 As shown in A, in the absence of extracellular Ca 2+ (0Ca 2+ ), 100 μM 4-chloro-3-ethylphenol induced ER / Ca2+ is released into the cytoplasm, but extracellular Ca 2+ Restore to 2mM (2Ca 2+ ) due to Ca 2+ Entering through SOC, it cannot induce [Ca 2+ ] cyt Further increase.

[0063] from Figure 2 As shown in B and 2C, dantrolene (10 μM), a selective inhibitor of RyR1 / 3, significantly attenuated the Ca2+-induced 2+ Release, but Ca 2+ did not enter HUVEC cells, which strongly suggests that 4-chloro-3-ethylphenol induced Ca2+ in the ER. 2+ The endothelial effect of 4-chloro-3-ethylphenol was confirmed at the cellular level by release through RyR3 but not by intracellular entry through SOC.

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

[0065] 1. Experimental materials:

[0066] LPS (Lipopolysaccharide) and GSK7975A were purchased from MCE Company; Elisa kits for detecting inflammatory factors were purchased from R&D Company in the United States.

[0067] 2. Experimental methods:

[0068] (1) HUVECs were cultured in a 12-well plate (density 5 × 105 cells / well) and the next step was performed after they were completely attached to the plate;

[0069] (2) Divided 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, LPS + 100 μM 4-chloro-3-ethylphenol + 10 μM GSK7975A group, cultured for 24 h;

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

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

[0072] 3. Analyze the data:

[0073] from Figure 3It can be seen that the levels of the three inflammatory factors in the model group were significantly increased compared with the control group, indicating that the HUVEC inflammation model was successfully established; compared with the model group, 4-chloro-3-ethylphenol significantly reduced the levels of inflammatory factors in the supernatant of the LPS-induced HUVEC cell inflammation model ( Figure 3 AC), indicating its anti-inflammatory effect, but this effect can be significantly inhibited by dantrolene, a selective inhibitor of RyR1 / 3, while the SOC inhibitor GSK7975A has no effect ( Figure 3 DF).

[0074] Example 3: Experimental study 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 as follows: 2.5 g DSS was dissolved in 100 mL deionized water.

[0077] 2. Experimental methods:

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

[0079] The 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 DSS solution, each group of mice was treated with drugs at the corresponding dose points, that is, the mice in the control group were given free drinking water and diet, and were gavaged with normal saline; the mice in the model group were given a solution containing 2.5% DSS to drink freely and diet, and were gavaged with normal saline; the mice in the experimental group 1 were given 2.5% DSS solution to drink freely, and 4-chloro-3-ethylphenol (5 mg / kg / day) was gavaged 7 days before DSS was given; the mice in the experimental group 2 were given 2.5% DSS solution to drink freely, and 4-chloro-3-ethylphenol (5 mg / kg / day) and dantrolene (5 mg / kg / day) were gavaged 7 days before DSS was given; the mice in the experimental group 3 were given 2.5% DSS solution to drink freely, and dantrolene (5 mg / kg / day) was gavaged 7 days before DSS was given;

[0080] (2) At the end of the experiment, mice were killed by cervical dislocation and laparotomy was performed to measure colon length; the results are shown in Figure 4 In the above statistical analysis figures, significant differences are expressed as * P<0.05, ** P<0.01, *** P < 0.001, ****P < 0.0001, ns means no statistical difference.

[0081] 3. Analyze the data:

[0082] The statistical results are as follows Figure 4 As shown in . Figure 4 A shows that the weight of mice in experimental group 1 was significantly higher than that in the model group, indicating that 4-chloro-3-ethylphenol can improve the weight of mice with ulcerative colitis. Figure 4 B shows 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. Figure 4 C shows that the colon length of mice in experimental group 1 is longer than that of mice in the model group and close to that of the control group, indicating that 4-chloro-3-ethylphenol can restore the colon length of mice with ulcerative colitis; Figure 4 The comparison between experimental groups 2 and 3 showed that dantrolene could reverse the therapeutic effect of experimental group 1, while dantrolene itself had no effect, indicating that 4-chloro-3-ethylphenol may stimulate RyR3 / ER / Ca 2+ Release pathway improves ulcerative colitis.

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

[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 5 mg / kg / d for five days), with 20 mice in each group.

[0086] 2. Experimental methods:

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

[0088] (2) CLP was performed on the model group and the drug pretreatment group to construct a sepsis model. The specific process is as follows:

[0089] a. Mice were fasted for 12 hours before the experiment;

[0090] b. After anesthesia, the animal was fixed on the operating board in supine position. The abdominal surgical area was routinely disinfected and hair removed. A 2 cm long incision was made on the abdominal wall with a scalpel under sterile conditions. The cecum was separated at the distal end of the ileocecal valve through the incision and ligated with No. 3 silk thread at 1 / 3 of the cecum;

[0091] c. Use an 18-gauge injection needle to pierce the ligated end and squeeze out a small amount of feces, trying to avoid damaging blood vessels. Then use No. 4 silk thread to suture the peritoneum and skin intermittently, and immediately inject 50 mL / kg body weight of normal saline subcutaneously to prevent shock. The sham operation group only opened the abdominal cavity, but did not perform ligation and puncture of the cecum.

[0092] (3) The death of mice was observed at 12h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h after surgery, and the survival curve was drawn. Figure 5 As shown, significant differences were used * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001, ns means no statistical difference.

[0093] 3. Analyze the data:

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

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

[0096] 1. Experimental materials:

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

[0098] 2. Experimental animals:

[0099] The mouse grouping, drug treatment and CLP modeling procedures were the same as those in Example 4.

[0100] 3. Experimental methods:

[0101] (1) 12 hours after CLP modeling, the dilation capacity of mouse mesenteric artery microvessels was measured:

[0102] a. The mice were killed by cervical dislocation, and the abdomen was placed upward on the operating table. The abdominal wall was opened and the mesenteric artery was isolated.

[0103] b. Slowly insert the first silver wire into the artery under a microscope and place it together with the blood vessel in another dish for later use;

[0104] c. Use microscopic tweezers and a small screwdriver to fix the two ends of the silver wire on one end of the detection clamp; bring the two measuring clamps close together, clamp the second silver wire with fiber tweezers, and insert it parallel to and close to the first silver wire. After the second silver wire is completely inserted, move it closer to the measuring clamp, fix the silver wire at the other end of the measuring clamp, and slightly separate the clamps;

[0105] d. After 20 minutes of heating and ventilation equilibrium, norepinephrine was added to constrict the blood vessels;

[0106] e. Add ACh / PSS from low to high concentration (the concentration gradient of ACh in the solution is 10 -8 -10 - 3 mol / L), to detect the tension after vascular dilation.

[0107] (2) Statistical results are as follows Figure 6 As shown in , significant differences were * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001, ns means no statistical difference.

[0108] 4. Analyze the data:

[0109] like Figure 6 As shown in AC, ACh-induced vascular relaxation neurotransmitter in the vagus nerve of CLP-septic mice was mostly impaired, and 4-chloro-3-ethylphenol significantly restored the impaired vascular relaxation. Figure 6 DF shows that in NO and PGI 2 In the presence of inhibitors L-NNA and indomethacin, 4-chloro-3-ethylphenol can also restore impaired vascular relaxation. At the same time, by comparing the maximum vascular relaxation percentage and area under the curve of the sham group, model group and drug pretreatment group, it was seen that the vascular relaxation capacity of the drug pretreatment group was significantly improved compared with the model group. These data indicate that 4-chloro-3-ethylphenol prevents sepsis by rescuing the EDH-mediated vascular relaxation 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 mouse grouping, drug treatment and CLP modeling procedures were the same as those in Example 4.

[0113] 2. Experimental methods:

[0114] (1) 12 hours after CLP modeling, mice were killed by cervical dislocation, eyeballs were removed to collect blood, and serum was collected after blood coagulation;

[0115] (2) According to the instructions of the Elisa kit, the levels of serum inflammatory factors in each group of mice were detected;

[0116] (3) Statistical results are as follows Figure 7 As shown in , significant differences were * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001, ns means no statistical difference.

[0117] 3. Analyze the data:

[0118] from Figure 7 It can be seen that the levels of proinflammatory factors TNF-α, IL-6, and IL-1β in the serum of the model group were significantly higher than those in the sham operation group; the levels of proinflammatory factors TNF-α, IL-6, and IL-1β in the serum of mice in the drug pretreatment group did not increase as much as those in the model group. This indicates that 4-chloro-3-ethylphenol can reduce the levels of proinflammatory 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: Effect of 4-chloro-3-ethylphenol on the survival of CLP-induced septic mice

[0120] 1. Experimental animals:

[0121] One hundred C57BL / 6 mice weighing 21-25 g were randomly divided into sham operation group, model group, 3 h postoperative drug administration group, 6 h postoperative drug administration group, and 12 h postoperative drug administration group, with 20 mice in each group.

[0122] 2. Experimental methods:

[0123] (1) CLP was performed on the model group and the groups given medication 3 h after surgery, 6 h after surgery, and 12 h after surgery to construct a sepsis model. The model construction process was the same as in Example 4. The sham operation group only opened the abdominal cavity, but did not perform cecal ligation and puncture.

[0124] (2) The groups receiving treatment 3 h after surgery, 6 h after surgery, and 12 h after surgery were given intragastric administration of 4-chloro-3-ethylphenol at a dose of 0.5 mg / kg at 3 h, 6 h, and 12 h after surgery, respectively;

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

[0126] (4) The results are as follows Figure 8 As shown, significant differences were used * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001, ns means no statistical difference.

[0127] 3. Analyze the data:

[0128] from Figure 8 It can be seen that no mice in the sham operation group died within 7 days; mice in the model group died 12 hours after modeling, and the survival rate was less than 20% 7 days after modeling. Compared with the model group, the 7-day survival rate of the group given medication 3 hours after surgery and the group given medication 6 hours after surgery was significantly higher than that of the model group, indicating that 4-chloro-3-ethylphenol has a good therapeutic effect on sepsis in mice.

[0129] Example 8: Effect of 4-chloro-3-ethylphenol on vasodilation in CLP-induced septic mice

[0130] 1. Experimental animals:

[0131] The mouse grouping, drug treatment and CLP modeling procedures were the same as in Example 7.

[0132] 2. Experimental methods:

[0133] (1) 24 hours after CLP modeling, the dilation capacity of mesenteric artery microvessels was measured. The specific experimental steps were the same as those in step 3(1)ae of Example 5;

[0134] (2) Statistical results are as follows Fig. 9 As shown in , significant differences were * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001, ns means no statistical difference.

[0135] 3. Analyze the data:

[0136] from Fig. 9 As can be seen from A, 9D and 9G, the vasodilation percentage of the model group was only about 20%, indicating that the vasodilation ability of the model group was severely impaired; Fig. 9 B and C), 6h postoperative medication group ( Fig. 9 E and F) and the group given medication 12 hours after surgery ( Fig. 9The percentage of vasodilation of H and I) increased with the increase of ACh concentration. The vasodilation capacity of the groups administered 3h, 6h and 12h after surgery was significantly improved compared with the model group. The above results show that the administration of 4-chloro-3-ethylphenol within 12h after modeling can effectively improve EDH-mediated vascular relaxation in septic mice and restore blood perfusion of multiple organs to treat sepsis in mice.

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

[0138] 1. Experimental animals:

[0139] The mouse grouping, drug treatment and CLP modeling procedures were the same as in Example 7.

[0140] 2. Experimental methods:

[0141] (1) 24 hours after CLP modeling, mice were killed by cervical dislocation, eyeballs were removed to collect blood, and serum was collected after blood coagulation;

[0142] (2) According to the instructions of the Elisa kit, the levels of serum inflammatory factors in each group of mice were detected;

[0143] (3) Statistical results are as follows Fig.10 As shown in , significant differences were * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001, ns means no statistical difference.

[0144] 3. Analyze the data:

[0145] from Fig.10 It can be seen that the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in the serum of the model group were significantly higher than those in the sham operation group. The levels of proinflammatory factors IL-1β and TNF-α in the serum of mice in the 3h and 6h postoperative administration groups were not as high as those in the model group; the level of proinflammatory factor IL-6 in the serum of the 3h postoperative administration group was not as high as that in the model group. The results show that 4-chloro-3-ethylphenol can significantly reduce the levels of proinflammatory factors IL-1β, IL-6, and TNF-α in the serum caused by sepsis. At the same time, the administration of 4-chloro-3-ethylphenol within 6h after modeling can significantly control the inflammatory response caused by sepsis and treat sepsis.

[0146] Example 10: Effect of 4-chloro-3-ethylphenol on liver and kidney function in CLP-induced septic mice

[0147] 1. Experimental materials:

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

[0149] 2. Experimental animals:

[0150] The mouse grouping, drug treatment and CLP modeling procedures were the same as in Example 7.

[0151] 3. Experimental methods:

[0152] (1) 24 hours after CLP modeling, mice were killed by cervical dislocation, eyeballs were removed to collect blood, and serum was collected after blood coagulation;

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

[0154] (3) Statistical results are as follows Fig.11 As shown in , significant differences were * P<0.05, ** P<0.01, *** P < 0.001, **** P < 0.0001, ns means no statistical difference.

[0155] 4. Analyze the data:

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

[0157] In summary, the present invention 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 Ca2+ Entering through SOC has a dual role. Further explanation, pure endothelial cell RyR / ER / Ca 2+ Release is a unique pathway that regulates EDH-mediated arterial vasodilation in health and has anti-inflammatory effects in ulcerative colitis and sepsis.

[0158] Compared with the prior art, the present invention discloses the following points for the first time: 1) In a healthy state, only endothelial cell ER / Ca 2+ Release may induce pure EDH-mediated arterial vasodilation; 2) Endothelial cell RyR / ER / Ca 2+ Release may have anti-inflammatory effects on ulcerative colitis and sepsis; 3) RyR / ER / Ca 2+ / EDH-mediated vasodilation may be a "friend" pathway for the prevention / treatment of ulcerative colitis and sepsis, but Ca entry through SOC 2+ May be an “enemy” pathway that worsens sepsis.

[0159] By using an animal model, the present invention discloses for the first time 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 regulates vascular endothelial cell calcium signals, reduces the level of inflammatory factors, and accelerates the repair of intestinal mucosa after damage, thereby effectively preventing and treating and alleviating ulcerative colitis; 2) 4-chloro-3-ethylphenol can maintain the body weight of mice with ulcerative colitis, restore their colon length, and improve diarrhea and bloody stools; 3) In a CLP-induced sepsis mouse model, it is confirmed that 4-chloro-3-ethylphenol can significantly reduce the mortality of septic mice, reduce proinflammatory factors, improve multiple organ dysfunction, prevent and treat sepsis by improving vasodilation of damaged blood vessels through EDH, and significantly prevent or treat liver and kidney function damage caused by sepsis.

[0160] We provide strong evidence that 4-chloro-3-ethylphenol has dual cellular targets, which may be superior to other single-target drugs such as those that inhibit only ER / Ca 2+ Dantrolene release and blockade of Ca 2+ Therefore, 4-chloro-3-ethylphenol, as a safe drug for the diagnosis of malignant hyperthermia, can be used as a potential new drug through the new RyR3 / ER / Ca 2+ release pathways for repurposing as new therapeutic agents for the prevention / treatment of ulcerative colitis and sepsis.

[0161] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should 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 drugs for preventing or treating ulcerative colitis.

2. The use of 4-chloro-3-ethylphenol according to claim 1 in the preparation of a drug for preventing or treating ulcerative colitis, characterized in that: 4-Chloro-3-ethylphenol is used to reduce the expression 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 drug for preventing or treating ulcerative colitis, characterized in that: 4-Chloro-3-ethylphenol is used to maintain the 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 diarrhea and blood in the stool in patients with ulcerative colitis.

4. Use of 4-chloro-3-ethylphenol in the preparation of drugs 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 survival in 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 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: Sepsis is caused by a bacterial or inflammatory infection resulting from one of the following conditions: pneumonia, cholangitis, peritonitis, urinary tract infection, abscess, or severe trauma.

9. Use of 4-chloro-3-ethylphenol in the preparation of a drug 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 damage and relieve organ dysfunction.

10. Use of 4-chloro-3-ethylphenol in the preparation of RyR receptor agonist drugs.

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