Application of vitamin B6 in preparation of medicine for preventing and / or treating NLRP6 inflammasome over-activated state diseases

Abstract

The invention belongs to the field of biomedicine, and particularly relates to application of vitamin B6 in preparation of drugs or preparations for preventing and / or treating NLRP6 inflammasome related diseases. The invention finds that vitamin B6 can be used for preparing medicines or preparations for preventing and / or treating NLRP6 inflammasome-related diseases by researching a specific mechanism that vitamin B6 inhibits excessive activation of NLRP6 inflammasome so as to treat NLRP6-related diseases. Compared with a traditional method (IL-18 binding protein, classic drugs for treating IBD, poor targeting property and non-response of part of people), the vitamin B6 can achieve good prevention and treatment effects only by oral administration. Vitamin B6 is low in cost and good in targeting property, can specifically inhibit NLRP6 expression, and has stronger targeting property than other enteritis treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of vitamin B6 in the preparation of drugs or preparations for preventing and / or treating NLRP6 inflammasome-related diseases. Background Art

[0002] Inflammasome is a protein complex that can act as a sensor of innate immunity and recognize a variety of signal stimuli. Inflammasomes are mainly composed of three parts: receptor protein, adaptor protein ASC and downstream caspase family. According to the different receptor proteins, inflammasomes can be divided into PYHIN family, such as AIM2 and IFI16, and the more widely studied NOD-like receptor (NLR) family, such as NLRP1, NLRP2, NLRP3, NLRC4, NLRP6, etc.

[0003] Nucleotide-binding oligomerization domain-like receptor 6 (NOD-like receptor family pyrin domain containing 6, NLRP6) is a member of the NLR family. Similar to other members of the NLR family, the NLRP6 structure includes an N-terminal furan domain (PYD), a central NLR family common structure NATCH domain, and a C-terminal leucine repeat sequence (LRR). NLRP6 is highly expressed mainly in gastrointestinal tissues, making the gastrointestinal tract the most widely studied organ for NLRP6. Activated NLRP6 forms self-oligomerization through the PYD and NATCH domains, providing a platform for the recruitment of the adaptor protein ASC. ASC further recruits the assembly and activation of downstream Caspase-1 to form a micron-scale inflammasome complex, which participates in the processing and maturation of downstream IL-18 and Gasdermin D, thereby mediating cell pyroptosis. NLRP6 is also involved in the regulation of NF-κB and MAPK signaling and the antibacterial and antiviral effector functions of the gastrointestinal tract. In addition, NLRP6 helps regulate mucus secretion and the production of antimicrobial peptides, thereby affecting intestinal microbial colonization and microbial-related infections, autoimmunity, metabolic diseases, and tumor diseases. NLRP6 has been found to be associated with the pathogenesis of a variety of intestinal diseases, such as inflammatory bowel disease (IBD), colorectal cancer (CRC), intestinal distress syndrome (IBS), etc.

[0004] Vitamin B6 (VB6) is a water-soluble vitamin, including three precursors that can be converted into each other in the body: pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM), as well as their corresponding phosphorylated derivatives: pyridoxine 5'-phosphate (PNP), pyridoxal 5'-phosphate (PLP), and pyridoxamine 5'-phosphate (PMP). The most active biological form is pyridoxal phosphate PLP. PLP is a coenzyme for more than 150 key enzymes in organisms and participates in many important biochemical processes in organisms, including amino acid metabolism, gluconeogenesis, lipid metabolism, and neurotransmitter biosynthesis. In addition to its function as a coenzyme, vitamin B6 has also been reported to have the functions of scavenging reactive oxygen, chelating metal ions, and promoting protein folding.

[0005] Vitamin B6 is widely present in the diet in the form of pyridoxine, pyridoxal and pyridoxamine, such as various meats, beans, nuts and dairy products, so severe VB6 deficiency is very rare. However, due to insufficient intake, poor absorption, disease background or drug inhibition, marginal VB6 deficiency symptoms are more common. Epidemiological surveys have shown that low PLP levels in the circulatory system are usually negatively correlated with the inflammatory marker C-reactive protein (CRP) and are associated with the progression of a variety of inflammatory diseases, such as rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Saibeni et al. reported that the plasma PLP content of IBD patients was significantly lower than that of the control group. In addition, low VB6 levels in the body are also associated with cardiovascular and cerebrovascular diseases (such as stroke, thrombosis, etc.), cancers (gastric cancer, colorectal cancer, kidney cancer, liver cancer, breast cancer, etc.), metabolic diseases (type 2 diabetes, etc.) and nervous system lesions (Parkinson's disease, Alzheimer's disease, etc.).

[0006] As an innate immune system sensor, NLRP6 inflammasome is highly expressed in intestinal tissues and plays a key role in inflammatory bowel disease, maintenance of intestinal microbial homeostasis and antiviral immune response. NLRP6 is widely involved in the occurrence of intestinal inflammatory diseases, so small molecule inhibitors that specifically target NLRP6 may provide new treatment ideas for such diseases. Summary of the invention

[0007] For inflammatory diseases caused by NLRP6 inflammasomes, the present invention found that vitamin B6 directly binds to and inhibits the activation of NLRP6 inflammasomes, and achieves the treatment of NLRP6 inflammasome-related diseases by regulating the body's IL-18 level. Specifically, the active form of vitamin B6, pyridoxal phosphate PLP, directly binds to the PYD domain of NLRP6, inhibits the interaction with the downstream adaptor protein ASC, thereby blocking the activation of NLRP6 inflammasomes and the release of downstream inflammatory cytokines.

[0008] The present invention firstly discloses the application of vitamin B6 in the preparation of IL-18 inhibitors, and achieves the treatment of NLRP6 inflammasome-related diseases by regulating the body's IL-18 level.

[0009] The present invention further discovered that PLP can significantly inhibit the activation of NLRP6 inflammasome both in vitro and in vivo, and was effectively verified in the TNF△ARE mouse spontaneous enteritis model and the DSS-induced mouse colitis model.

[0010] The second aspect of the present invention discloses the use of vitamin B6 in the preparation of drugs for preventing and / or treating diseases caused by NLRP6 inflammasomes.

[0011] Among them, vitamin B6 can be prepared as an NLRP6 inhibitor.

[0012] Preferably, the vitamin B6 is a combination of one or more of pyridoxine, pyridoxal, pyridoxamine, pyridoxine phosphate, pyridoxal phosphate or pyridoxamine phosphate.

[0013] More preferably, the vitamin B6 is pyridoxal phosphate.

[0014] The diseases induced by NLRP6 inflammasome are specifically inflammatory diseases mediated by abnormal activation of NLRP6 inflammasome, including inflammatory bowel disease, enterovirus and bacterial infection, dysbiosis, colitis-related carcinogenesis, congenital megacolon, irritable bowel syndrome, rheumatoid arthritis, pulpitis and apical periodontitis.

[0015] In a specific embodiment, abnormal activation of NLRP6 inflammasome refers to a detection value of IL-18 secretion in human serum that is higher than the median value of normal people.

[0016] In a specific embodiment, abnormal activation of NLRP6 inflammasome refers to a detection value of IL-18 secretion in human serum that is higher than 1500 pg / mL.

[0017] Inflammatory bowel disease (IBD) refers to a group of nonspecific chronic gastrointestinal inflammatory diseases of unknown cause, including ulcerative colitis (UC), Crohn's disease (CD) and indeterminate colitis (IC).

[0018] Enterovirus infection includes viral infections caused by 71 serotypes of enterovirus, including poliovirus, Coxsackievirus, enterocytopathic human orphan virus (ECHO, abbreviated as echovirus) and new enterovirus.

[0019] The preferred medicine also includes a pharmaceutically acceptable carrier and adjuvant. The medicine can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions.

[0020] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.

[0021] The third aspect of the present invention discloses the use of the above-mentioned drug in combination with one or more of the following in the preparation of a drug for NLRP6 inflammasome-induced diseases;

[0022] (1) agents that increase the efficacy of vitamin B6 on IL-18 inhibitors;

[0023] (2) agents that increase the inhibitory efficacy of vitamin B6 on NLRP6;

[0024] (3) Agents that enhance the ability of vitamin B6 to target the PYD domain of NLRP6.

[0025] The fourth aspect of the present invention discloses the use of NLRP6 inhibitors in the preparation of drugs for preventing and / or treating diseases caused by overexpression of TNFα.

[0026] The NLRP6 inhibitor is vitamin B6.

[0027] The fifth aspect of the present invention discloses a method for preparing an animal model of NLRP6 overactivation, the method comprising feeding an animal with a vitamin B6-deficient food for at least 21 days, thereby obtaining an animal model of NLRP6 overactivation.

[0028] The method optionally includes detecting overactivation of NLRP6 in the animal after feeding the animal a vitamin B6-deficient diet for at least 21 days.

[0029] The animal model prepared by the method can be various animal models. In a specific preferred embodiment, the animal is a rodent, preferably a rat or a mouse, and more preferably a mouse.

[0030] The sixth aspect of the present invention discloses the use of the NLRP6 over-activated animal model obtained by the above-mentioned preparation method in screening drugs for preventing and / or treating diseases caused by NLRP6 inflammasome.

[0031] The NLRP6 inflammasome-induced diseases are inflammatory diseases mediated by abnormal activation of the NLRP6 inflammasome, including but not limited to inflammatory bowel disease, enterovirus and bacterial infections, dysbiosis, colitis-related carcinogenesis, congenital megacolon, irritable bowel syndrome, rheumatoid arthritis, pulpitis and apical periodontitis.

[0032] The abnormal activation of NLRP6 inflammasome refers to the detection value of IL-18 secretion in human serum being higher than the median of normal people.

[0033] In a specific embodiment, abnormal activation of NLRP6 inflammasome refers to a detection value of IL-18 secretion in human serum that is higher than 1500 pg / mL.

[0034] The present invention achieves the following beneficial technical effects:

[0035] Currently, there is no research on the role of vitamin B6 in regulating inflammatory bowel disease (IBD) and other NLRP6 inflammasome-related diseases. This application studies the specific mechanism by which vitamin B6 inhibits excessive activation of NLRP6 inflammasomes and thus treats NLRP6-related diseases, and finds that vitamin B6 can be used to prepare drugs or preparations for preventing and / or treating NLRP6 inflammasome-related diseases.

[0036] At the same time, compared with traditional methods (IL-18 binding protein, a classic drug for treating IBD, which is not very targeted and some people do not respond), vitamin B6, as a trace element, has no reported side effects on the human body at conventional doses, and can achieve good prevention and treatment effects simply through oral administration.

[0037] Vitamin B6 is low-cost, has good targeting, and can specifically inhibit the expression of NLRP6. Because NLRP6 is specifically highly expressed in the intestine, it is more targeted than other enteritis treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results show that vitamin B6 inhibits the activation of NLRP6 inflammasome in vivo under steady-state conditions. Figure 1 a shows the feeding treatment method of mice, Figure 1 b shows the GSDMD shearing status detected by Western Blot in mouse jejunum tissue. Figure 1 c shows the level of interleukin 18 (IL-18) in mouse intestinal tissue. It can be seen that when vitamin B6 is deficient, NLRP6 inflammasome activation is significantly enhanced, and its downstream GSDMD shearing and IL-18 release are significantly enhanced.

[0039] Figure 2 The binding of vitamin B6 to NLRP6 protein is shown. Figure 2 a shows the size and purity of purified proteins (including full-length protein MBP-mNLRP6-GFP, as well as truncated proteins MBP-mNLRP6PYD-GFP and MBP-mNLRP6LRR-GFP) detected by Coomassie Brilliant Blue staining. All proteins will be used in subsequent in vitro binding assays. Figure 2 b-2f show the microcalorimetry detection of the binding of the full-length protein MBP-mNLRP6-GFP with different forms of VB6, PLP, PL, PM, PMP, and PN. It can be seen that only the active form of VB6, PLP, directly binds to the NLRP6 protein, and the equilibrium dissociation constant Kd value is 143nM. Figure 2 g-2i shows the binding of NLRP6 domain truncated proteins MBP-mNLRP6△NATCH-GFP (NLRP6 NATCH domain deleted), MBP-mNLRP6PYD-GFP (only NLRP6 PYD domain), MBP-mNLRP6LRR-GFP (only NLRP6 LRR domain) to PLP by microcalorimetry. It can be seen that the PYD domain of NLRP6 can directly bind to PLP, and the equilibrium dissociation constant Kd value is 126nM. However, PLP has no direct binding to the LRR domain of NLRP6, and the deletion of the NATCH domain of NLRP6 has no significant effect on the binding of NLRP6 to PLP, proving that NLRP6 binds to PLP through the PYD domain.

[0040] Figure 3 The mechanism by which PLP, the active form of VB6, inhibits NLRP6 inflammasome activation is shown. Figure 3a shows the effect of PLP on the binding of NLRP6 and ASC detected by immunoprecipitation, and it can be seen that PLP significantly inhibits the binding of NLRP6 and ASC. Figure 3 b-3c shows the in vitro reconstruction of NLRP6 inflammasome, and the effect of PLP on downstream indicators of NLRP6 inflammasome activation, such as ASC oligomerization, Caspase-1 cleavage, and GSDMD cleavage, was detected by WB. It can be seen that PLP significantly inhibited the activation of NLRP6 inflammasome. Figure 3 d-3e shows the effect of PLP on NLRP6 inflammasome detected by immunofluorescence, which shows that PLP significantly inhibits the co-localization of NLRP6 and the downstream adaptor protein ASC. Figure 3 f-3h shows the live cell imaging of in vitro reconstructed NLRP6 inflammasome, and the level of cell pyroptosis was detected by PI staining and lactate dehydrogenase LDH release. It can be seen that PLP significantly inhibits cell pyroptosis mediated by NLRP6 inflammasome activation.

[0041] Figure 4 Shown is the effect of vitamin B6 deficiency on spontaneous intestinal inflammation in TNFΔARE mice. Figure 4 a shows the treatment process of the TNFΔARE mouse spontaneous intestinal inflammation model. Figure 4 b shows the GSDMD shearing status detected by Western Blot in mouse jejunum and ileum tissues. Figure 4 c-4d shows the levels of interleukin 18 (IL-18) in the intestinal tissue and serum of mice, respectively. It can be seen that when vitamin B6 is deficient, the activation of NLRP6 inflammasome in TNFΔARE enteritis mice is significantly enhanced, and its downstream GSDMD shearing and IL-18 release are significantly enhanced, indicating that the inflammation in the mice is more severe.

[0042] Figure 5 Shown is the effect of vitamin B6 deficiency on DSS-mediated colitis in normal mice. Figure 5 a shows the process of inducing and treating the DSS-mediated enteritis model in normal mice. Figure 5 b shows the body weight changes in the mouse DSS colitis model. Figure 5 c shows the diarrhea score of the mouse DSS colitis model. Figure 5 d shows the survival rate of mice. Figure 5 e-5f shows the colon length and statistical analysis of the mouse DSS colitis model. It can be seen that the VB6-deficient group had more severe enteritis symptoms than the control group, as shown by significantly reduced body weight, higher diarrhea scores, shorter colon length, and lower survival rate. This proves that vitamin B6 deficiency significantly aggravates DSS-mediated colitis in normal mice.

[0043] Figure 6Shown is the effect of vitamin B6 deficiency on DSS-mediated colitis in NLRP6 knockout mice. Figure 6 a shows the process of induction and treatment of DSS-mediated enteritis model in NLRP6 knockout mice. Figure 6 b shows the body weight changes in the mouse DSS colitis model. Figure 6 c shows the diarrhea score of the mouse DSS colitis model. Figure 6 d-6e shows the colon length and statistical analysis of the mouse DSS colitis model. It can be seen that there is no significant difference in the symptoms of enteritis between the VB6-deficient group and the control group, and there is no significant difference in weight change, diarrhea score, colon length, etc. This proves that vitamin B6 deficiency has no significant effect on inflammatory bowel disease in NLRP6 knockout mice, further proving that vitamin B6 exerts its effect by inhibiting the NLRP6 pathway.

[0044] Figure 7 Oral administration of vitamin B6 improves inflammatory bowel disease in mice. Figure 7 a shows the process of inducing the mouse DSS-mediated enteritis model. Figure 7 b shows the body weight changes in the mouse DSS colitis model. Figure 7 c shows the diarrhea score of the mouse DSS colitis model. Figure 7 d-7e shows the colon length and statistical analysis of the mouse DSS colitis model. Figure 7 f-7g shows the H&E staining of the mouse colon tissue pathological sections. It can be seen that the VB6-added group had milder enteritis symptoms than the control group, as shown by a significant increase in body weight, lower diarrhea score, longer colon length, and lower tissue damage score. This proves that oral VB6 can significantly improve the DSS-induced enteritis phenotype in mice.

[0045] Figure 8 Shown is the improvement of inflammatory bowel inflammation in mice by intravenous administration of vitamin B6. Figure 8 a shows the process of inducing the mouse DSS-mediated enteritis model. Figure 8 b shows the body weight changes in the mouse DSS colitis model. Figure 8 c shows the diarrhea score of the mouse DSS colitis model. Figure 8 d-8e show the colon length and statistical analysis of the mouse DSS colitis model. Figure 8 f-8g shows the pathological sections of the mouse colon tissue. It can be seen that the VB6-added group had milder enteritis symptoms than the control group, as shown by a significant increase in body weight, lower diarrhea score, longer colon length, and lower tissue damage score. This proves that intravenous injection of VB6 can significantly improve the DSS-induced enteritis phenotype in mice.

[0046] Fig. 9 It was shown that the major source of IL-18 in the intestine is NLRP6-mediated secretion.

[0047] Fig.10 It was shown that IL-18 was significantly elevated in IBD patients.

[0048] Fig.11 A clinical study protocol for the treatment of patients with inflammatory bowel disease with oral vitamin B6 is shown.

[0049] Fig.12 The changes in Mayo scores, simplified CDAI scores, and clinical remission rates of IBD patients in stages V1 and V5 after oral vitamin B6 administration are shown, as well as serum PLP levels in patients in the V1 baseline period detected by mass spectrometry and serum IL-18 levels in patients in the V1 baseline period detected by enzyme-linked immunosorbent assay (ELISA). DETAILED DESCRIPTION

[0050] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The raw materials and equipment used in the examples are well known to those skilled in the art, and are all available on the market or easily obtained or prepared.

[0051] The following examples used GraphPad8.0.2 statistical software to process the data. The experimental results were expressed as mean ± standard deviation. The data were tested using ANOVA, and pairwise comparisons were performed using T test. Multiple groups were tested using Tukey's multiple comparison test. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated a very significant difference.

[0052] The C57BL / 6 mice in the following examples were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and the age of the mice in the experiments was 6-8 weeks.

[0053] Example 1 Vitamin B6 inhibits NLRP6 inflammasome activation in vivo under steady state conditions

[0054] 1. Experimental process:

[0055] The experimental mice (C57BL / 6 mice, 4 weeks old) were randomly divided into three groups: the first group was given vitamin B6-deficient food (customized vitamin B6-deficient feed, purchased from Trophy Company, Nantong, Jiangsu) and drinking water; the second group was given normal control food (AIN93G standard feed, purchased from Trophy Company, Nantong, Jiangsu) and drinking water; the third group was given normal control food (AIN93G standard feed, purchased from Trophy Company, Nantong, Jiangsu) and additional vitamin B6 (50 mg / kg / day) was added to the drinking water for feeding. After three weeks of feeding, the mice were euthanized and intestinal tissue samples were taken for Western Blot and ELISA analysis.

[0056] 2. Western Blot sample collection and processing method:

[0057] After the mice were killed by cervical dislocation, 0.5 cm of jejunal tissue was obtained, the contents were rinsed out, and then the tissue was cut open longitudinally and placed in pre-cooled 200 μL RIPA lysis buffer (with protein inhibitors PI and PMSF added at a ratio of 1:100), and 4-5 steel beads were added to thoroughly homogenize and disperse the cells. After lysis on ice for 15 min, the cells were centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant after centrifugation was added to 3× loading tube for mixing, and the samples were boiled at 100°C for 10 min for subsequent Western Blot detection.

[0058] 3. Specific process of Western Blot detection:

[0059] Place the prepared 10% gel plate in the electrophoresis instrument, and add 1x running buffer to the electrophoresis instrument. After checking for leakage, load the samples in the preset order. Turn on the power supply, the voltage corresponding to the concentrated gel is 80v, 30min, and the voltage corresponding to the separation gel is 120v, 1h. According to the size of the target protein, the voltage can be appropriately increased to adjust the gel running time. Soak the cut PVDF membrane in methanol for about 30s until it is translucent, open the transfer clamp, immerse the black side in Transfer, and place a filter paper soaked in Transfer on each of the black and white clamp surfaces; pry open the gel plate, cut off the concentrated gel, remove the separation gel from the long gel plate, and place the gel in the transfer clamp in the order of black gauze-filter paper-gel-PVDF membrane (about 9×5.6cm)-filter paper (about 10×8.5cm)-black gauze from bottom to top. Slightly fix the gel with your fingers, cover the activated PVDF membrane on the gel, drive out the bubbles between the membrane and the gel, and close the transfer clamp. Place the combined transfer clip in the electrophoresis tank, and place the red and black colors in the transfer tank accordingly. Place an ice box in the transfer tank and fill it with transfer buffer. Set the voltage to 90v and the current to about 300mA for 1.5h; after the transfer is completed, take out the PVDF membrane and transfer the PVDF membrane to 5% skim milk and block it on a shaker at room temperature for one hour; after blocking, rinse the PVDF membrane three times with PBST, and shake it at room temperature for 5min each time. Then place the PVDF membrane in a clean incubation box, place it on a shaker at 4℃, and incubate the primary antibody overnight; recover the primary antibody and wash the membrane three times with TBST, and shake it at room temperature for 10min each time. Add the secondary antibody configured at 1:5000 and shake it at room temperature for 30min; after the secondary antibody is blocked, wash the membrane 3 times with PBST, each time for 10min, then directly expose the PVDF membrane after reacting with the developing substrate, and develop it using a Biorad developer.

[0060] 4.ELISA sample collection and processing method:

[0061] After the mice were killed by cervical dislocation, 0.5 cm of jejunal tissue was obtained, the contents were rinsed out, and then the tissue was cut open longitudinally and placed in 500 ul DMEM culture medium. After culturing for 3 hours at 37°C and 5% CO2, the culture supernatant was obtained and centrifuged at 800 rpm for 5 minutes. The supernatant was aliquoted and stored at -40°C for subsequent ELISA testing.

[0062] 5. Specific process of ELISA detection of IL-18:

[0063] The mouse IL-18 ELISA kit used in the experiment was purchased from Invitrogen (Cat#: BMS618). The specific process is as follows: Dilute the Capture antibody with PBS to the corresponding concentration, add 50ul to each well of the ELISA plate (96-well), and incubate overnight at 4°C. Wash three times with PBST and remove the remaining liquid in the plate. Add 150ul of blocking solution (PBS containing 1% fetal bovine serum) to each well and incubate at room temperature for 30 minutes. Wash three times with PBST and remove the remaining liquid in the plate. Dilute the standard with PBS to the corresponding concentration, add 100ul of standard dilution or sample solution to the ELISA plate per well, and incubate at room temperature for 2 hours. Wash three times with PBST and remove the remaining liquid in the plate. Dilute the Detection antibody with PBS to the corresponding concentration, add 50ul to each well of the ELISA plate, and incubate at room temperature for 1 hour. Wash three times with PBST and remove the remaining liquid in the plate. Dilute HRP with PBS to the corresponding concentration, add 50ul to each well of the ELISA plate, and incubate at room temperature for 1 minute. Wash three times with PBST, remove the remaining liquid in the plate, add 50ul TMB substrate to each well, incubate at room temperature, and when the color development is appropriate, add 50ul sulfuric acid (1M) to each well to terminate the color development reaction, and immediately place in an ELISA reader for detection (450nm).

[0064] 6. Experimental results:

[0065] like Figure 1 b shows that the experimental results indicate that when mice were fed with food lacking vitamin B6, the GSDMD splicing downstream of the NLRP6 inflammasome was significantly enhanced compared with mice fed a normal diet or a diet supplemented with vitamin B6, indicating that NLRP6 inflammasome activation was significantly enhanced in the absence of VB6.

[0066] NLRP6 proteolysis activates Caspase-1 and promotes the maturation and release of the inflammatory cytokine interleukin 18 (IL-18). The function of NLRP6 in promoting epithelial cell repair depends on the production of IL-18, and it can promote the formation of the intestinal epithelial mucus layer through autophagy. In the state of intestinal inflammation, the increase in IL-18 secretion will lead to the loss of goblet cells, destroy the intestinal mucosal barrier and promote the aggravation of inflammation. Therefore, the degree of inflammation in the body is closely related to the level of IL-18.

[0067] The experimental results showed that the intestinal tissue of the VB6-deficient group released significantly more inflammatory cytokine IL-18 than the mice in the normal diet group and the vitamin B6-supplemented diet group ( Figure 1 c, *p<0.05, ***p<0.001), that is, VB6 inhibited the expression of IL-18. The above results all indicate that under steady-state conditions, vitamin B6 significantly inhibits the activation of NLRP6 inflammasomes in vivo.

[0068] Results and Discussion:

[0069] The experimental results of Example 1 showed that under steady-state conditions, NLRP6 inflammasomes were abnormally activated in mice deficient in vitamin B6, suggesting that vitamin B6 has a potential inhibitory effect on NLRP6 inflammasomes. We speculate that vitamin B6 is a targeted inhibitor of NLRP6 and further verify its molecular mechanism of inhibiting NLRP6 inflammasomes.

[0070] Example 2 The active form of vitamin B6, PLP, can directly bind to the NLRP6 PYD domain

[0071] 1. Cloning of eukaryotic protein expression vector:

[0072] Full-length protein MBP-mNLRP6-GFP, three truncated proteins MBP-mNLRP6 PYD -GFP, MBP-mNLRP6 LRR -GFP and MBP-mNLRP6 △NATCH -GFP were cloned into pFastBac-HTB vector to construct insect cell expression vector, which carried MBP tag, His tag, GFP fluorescent protein and TEV restriction site.

[0073] 2. Transformation of DH10Bac competent cells:

[0074] All insect cell expression vectors were transformed into Escherichia coli DH10Bac competent cells for the production of bacmids. Specifically, 100uL of DH10Bac competent cells were thawed on ice. 100ng of pFastBacHTA plasmid was added and incubated on ice for 30min. Heat shock was performed in a 42℃ water bath for 45s, and then immediately placed on ice for 2min. 900uL of LB liquid culture medium was added and cultured at 37℃ with shaking at 180rpm for 4h. 100uL of resuscitated cells were added with 100ug / mL Bluo-gal and 40ug / mL IPTG, and spread on LB plates containing 50ug / mL kanamycin, 7ug / mL gentamicin, and 10ug / mL tetracycline. After culturing at 37℃ for 48h, large, isolated white single colonies were selected for the next step of identification.

[0075] 3. Bacteria culture and collection:

[0076] Inoculate a correctly identified white spot colony into LB liquid culture medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline, and culture at 37°C with shaking overnight.

[0077] 4. Extraction and separation of recombinant bacmid DNA:

[0078] Take 5mL of bacteria and centrifuge at 14000g, 4℃, 1min to collect bacteria. Add P1 (300ul) to resuspend the bacteria, P2 (300ul) to lyse, and P3 (300ul) to neutralize, then centrifuge at 12000rpm for 10min, take 800ul of supernatant, add 640ul of isopropanol to mix, incubate at -20℃ for 4h, centrifuge at 14000rpm for 15min, remove the supernatant, add 70% ethanol to wash the precipitate twice, wash it once with 100% ethanol, dry it for half an hour, add 50ul ddH2O, take 1ul for PCR identification, and transfect the rest.

[0079] 5. Bacmid transfection and amplification of recombinant baculovirus:

[0080] SF9 cells were plated 1 hour in advance at a cell density of 4x10 5 / ml / well. Transfection system: 100ul Grace's Insectmedium plus 6ul bacmid, 100ul plus 6ul Cellfection II reagent, mix them separately, add them together and mix again, incubate for 30min. After the incubation, add 800ul Grace's medium, discard the medium in the 6-well plate, wash it with 2ml Grace's medium, and add the transfection system. Change the medium after 5h; collect the supernatant three days later as the first-generation recombinant baculovirus P1. Amplify the virus to increase the titer. When the virus is amplified to the third generation, infect SF9 cells in large quantities for protein expression. 400ml cells, 2% inoculated with the third-generation recombinant virus P3, collect the cells three days later, add 30ml lysis buffer to resuspend, ultrasonicate on ice for 20min, 50% power, 2s on, 2s off. Centrifuge at 20000rpm, 4℃ for 2h. After the supernatant passes through a 0.22um filter membrane, it is purified by Ni column, removed by 50mM imidazole, and eluted with 500mM imidazole. The eluted protein solution was collected and added to a gel filtration column to further purify the target protein. 10ul of the protein solution was taken for gel running and Coomassie Brilliant Blue staining was used to detect protein expression and purity. Specific cells and strains are shown in Table 1 below.

[0081] Table 1. Cells and strains

[0082] Cells and strains source Identification Code E.coli DH5-alpha New England Biolabs Cat#C2987 SF9 cells ThermoFisher (GIBCO) B82501 DH10Bac ThermoFisher (GIBCO) 10361-012

[0083] 6. Microscale thermophoresis (MST) detection of protein binding to VB6:

[0084] Add VB6 with a preset concentration gradient to the purified protein (200nM) and gently pipette to mix. Load the mixed liquid in the NanoTemper capillary and fix it to the detection plate of the Monolith NT.115 instrument to run the affinity detection program. Use NanoTemper software to analyze the data obtained and calculate the equilibrium dissociation constant (Kd).

[0085] 7. Experimental results:

[0086] Figure 2 The figure shows the binding of vitamin B6 to NLRP6 protein. Figure 2 a shows that the purified proteins (including the full-length protein MBP-mNLRP6-GFP and the truncated protein MBP-mNLRP6) were detected by Coomassie Brilliant Blue staining. PYD -GFP and MBP-mNLRP6 LRR -GFP) size and purity, the proteins were all of the correct size and well expressed, with high purity and no obvious mixed bands, and could be used for subsequent in vitro binding experiments.

[0087] Figure 2 b-2f respectively show the binding of the full-length protein MBP-mNLRP6-GFP to different forms of VB6, PLP, PL, PM, PMP, and PN, detected by microcalorimetry (MST). The equilibrium dissociation constant Kd value is inversely proportional to the affinity. It can be seen that only the active form of VB6, PLP, has an equilibrium dissociation constant Kd value of 143nM, which directly binds to the NLRP6 protein. Other forms of VB6 and NLRP6 are at the μM level, with almost no binding. Therefore, subsequent in vitro experiments were processed in the form of PLP.

[0088] The NLRP6 structure includes the N-terminal furan domain (PYD), the middle NLR family common structure NATCH domain, and the C-terminal leucine repeat sequence (LRR). We constructed truncated proteins with different domains to find the binding site of PLP and NLRP6. Figure 2 g-2i show the detection of NLRP6 domain truncated protein MBP-mNLRP6 by microcalorimetry. △NATCH -GFP (NATCH domain of NLRP6 deleted), MBP-mNLRP6 PYD -GFP (only the PYD domain of NLRP6), MBP-mNLRP6 LRR-GFP (only the LRR domain of NLRP6) binding to PLP shows that the PYD domain of NLRP6 can directly bind to PLP, and the equilibrium dissociation constant Kd value is 126nM. However, PLP has no direct binding to the LRR domain of NLRP6, and the affinity constant of the binding between the two cannot be fitted. In addition, the deletion of the NATCH domain of NLRP6 has no significant effect on the binding of NLRP6 to PLP, which is manifested in that the affinity is not significantly different from the full-length protein, proving that NLRP6 binds to PLP through the PYD domain.

[0089] Results and Discussion:

[0090] The experimental results of Example 2 showed that the active form of VB6, PLP, can directly bind to the NLRP6 PYD domain, providing strong evidence that VB6 is a targeted inhibitor of NLRP6. Next, we will use cell experiments to further verify the molecular mechanism by which VB6 inhibits NLRP6 inflammasome activation.

[0091] Example 3 Mechanism of Vitamin B6 Inhibiting NLRP6 Inflammasome Activation

[0092] 1. Co-immunoprecipitation (CO-IP)

[0093] Plate HEK-293T cells into 6-well plates with a cell density of 50%-60% and culture overnight. When the cell density is about 70%, transfect the corresponding plasmid. Pipette 100ul Opti-MEM medium into a 1.5ml sterile Ep tube, add an appropriate amount of plasmid, mix well, and let stand for 5 minutes; after standing, add the corresponding dose of transfection reagent PEI (the mass ratio of plasmid to PEI is 1:4), mix well, and let stand for 15 minutes. After standing, add the mixed liquid to the cell culture supernatant and shake gently to mix it. Change the medium after 4h and add 10uM PLP to treat the cells.

[0094] After 36 hours, harvest cells: discard the supernatant, wash the cells with pre-cooled PBS, add 400ul NP-40, and lyse for 30 minutes on a shaker at 4℃. Collect the cell lysate into a 1.5ml EP tube, centrifuge at 4℃, 12000rpm for 10 minutes. Take 40ul of cell lysate, add 20ul 3×Sample buffer, heat at 100℃ for 10 minutes, and use it as input for Western blot detection; transfer the remaining cell lysate to a new 1.5ml EP tube for immunoprecipitation experiments.

[0095] Add an appropriate amount of washed anti-Flag magnetic beads, incubate at 4℃ for 2 hours, and then centrifuge at 4℃ 3000rpm for 1min. Discard the supernatant, add 1mI NP-40 lysis buffer to resuspend the magnetic beads, and centrifuge at 4℃ 3000rpm for 1min. Repeat five times to fully wash the magnetic beads. Aspirate the supernatant thoroughly for the last time, add 60ul 1×Sample buffer, heat at 100℃ for 10min, and use it as IP sample for Western blot detection;

[0096] 2. In vitro NLRP6 inflammasome reconstruction and ASC aggregation detection:

[0097] HEK-293T cells were plated into 6-well plates at a cell density of 50%-60% and cultured overnight. When the cell density reached about 70%, plasmids were transfected. The following plasmids were required for the reconstruction of NLRP6 inflammasome in vitro: pCDNA FLAG-mNLRP6, pCDNAHA-mASC, plvx-Myc-mCasp1, pCDNA FLAG-mGSDMD. The medium was changed after 4 hours, and 0.1-100uM PLP was added to the cells in the treatment group.

[0098] After 24 hours, the cell supernatant was collected for the detection of LDH secretion level. The culture medium was added again, and the cells were stained with PI for live cell imaging to observe the level of cell pyroptosis. The cells were washed three times with pre-cooled PBS (1 ml per well), PBS was discarded, 60ul 1×Sample buffer was added, and heated at 100℃ for 10 minutes. The samples were used for Western blot to detect the activation of NLRP6 inflammasome;

[0099] ASC aggregation detection: Wash the cells three times with pre-cooled PBS (1 ml per well), discard PBS, add 200ul NP-40 to each well, and lyse on a shaker at 4℃ for 30min. Centrifuge at 4℃, 12000rpm for 10min. Take 40ul of cell lysate, add 20ul 3×Sample buffer, heat at 100℃ for 10min, and use it as input for Western blot detection; discard the remaining supernatant, and wash the precipitate three times with 1ml pre-cooled PBS. After centrifugation, discard the supernatant, resuspend the precipitate with 500ul pre-cooled PBS, add 2mM DSS (Disuccinimidylsuberate), and incubate at room temperature for 30min for cross-linking. After cross-linking, centrifuge at 4℃, 2000rpm for 15min, discard the supernatant, add 60ul 1×Sample buffer, heat at 100℃ for 10min, and use it for Western blot detection of ASC aggregation in the precipitate.

[0100] Specific Western Blot antibodies are shown in Table 2 below.

[0101] Table 2. Western Blot Antibodies

[0102]

[0103] 3. Experimental results:

[0104] Figure 3 The mechanism by which PLP, the active form of VB6, inhibits NLRP6 inflammasome activation is shown. Figure 3 a The effect of PLP on the binding of NLRP6 and ASC was detected by immunoprecipitation. It can be seen that the addition of PLP significantly inhibited the binding of NLRP6 and ASC, indicating that PLP inhibits inflammasome activation by blocking the binding of NLRP6 to the downstream adaptor protein ASC.

[0105] Figure 3 b-3c shows the in vitro reconstruction of NLRP6 inflammasomes, and the effect of PLP on downstream indicators of NLRP6 inflammasome activation, such as ASC oligomerization, Caspase-1 cleavage, and GSDMD cleavage, was detected by WB. The experimental results showed that PLP significantly inhibited the activation of NLRP6 inflammasomes, specifically, the addition of PLP significantly weakened the oligomerization of ASC, the cleavage of Caspase-1, and the cleavage of GSDMD, and these effects were all concentration gradient dependent.

[0106] Figure 3 d Laser confocal imaging showed the effect of PLP on the co-localization of NLRP6 and ASC. It can be seen that after the co-localization of NLRP6 and ASC in the normal group, obvious puncta were formed in the cells. The addition of PLP significantly inhibited the formation of co-localization puncta of NLRP6 and downstream adaptor protein ASC (***p<0.001, ****p<0.0001), and it was concentration gradient dependent.

[0107] Figure 3 f-3h shows live cell imaging of in vitro reconstructed NLRP6 inflammasomes, and the level of cell pyroptosis was detected by PI staining and lactate dehydrogenase LDH release. Obvious pyroptosis morphology was observed in the normal group cells, with a high proportion of PI staining positive and significant LDH release in the cell culture supernatant. After PLP treatment, cell pyroptosis was significantly reduced, the proportion of PI staining positive was significantly reduced, and the amount of LDH released in the cell culture supernatant was significantly reduced (**p<0.01, ***p<0.001, ****p<0.0001). The above results indicate that PLP significantly inhibits cell pyroptosis mediated by intracellular NLRP6 inflammasome activation.

[0108] Results and Discussion:

[0109] The experimental results of Example 3 further demonstrated the mechanism by which vitamin B6 inhibits the activation of NLRP6 inflammasomes, namely, PLP directly binds to NLRP6 protein, inhibiting its interaction with the downstream adaptor protein ASC, thereby blocking the oligomerization of downstream ASC, the cleavage of Caspase-1, and the cleavage of GSDMD, thereby inhibiting the occurrence of cell pyroptosis. We have demonstrated that VB6 can significantly inhibit the activation of NLRP6 inflammasomes in vitro, and next we further explored in vivo whether VB6 can play a role in inhibiting the excessive activation of NLRP6 inflammasomes.

[0110] Example 4 Vitamin B6 deficiency significantly promotes TNF ΔARE NLRP6 inflammasome activation in mice

[0111] 1. Experimental model:

[0112] TNF ΔARE The mouse model is a mutant mouse model that carries a deletion of an AU-rich element (ARE) in the 3'-uncoding region (3'-UTR) of the tumor necrosis factor α (TNFα) gene. This deletion enhances the stability of TNFα mRNA and systemically increases the production of its translation product, i.e., systemic TNFα overexpression, leading to progressive spontaneous arthritis and inflammatory bowel disease (IBD). As we age, TNFα is overexpressed. ΔARE The mice gradually develop spontaneous arthritis symptoms with joint swelling, hind limb deformity, and spontaneous inflammation of the entire intestine, accompanied by weight loss. The above symptoms begin to develop as early as 3 weeks of age and are generally fully developed after 10 weeks of age. This model is often used for observational studies related to the development / treatment of IBD and similar diseases. A large number of studies have shown that TNF ΔARE The mouse model is very similar to human Crohn's disease (CD).

[0113] 2. Experimental process:

[0114] Experimental mice (TNF △ARE Mice, 4 weeks old) were randomly divided into two groups: the first group was given vitamin B6-deficient food (customized vitamin B6-deficient feed, purchased from Trophy Company, Nantong, Jiangsu) and drinking water; the second group was given normal control food (AIN93G standard feed, purchased from Trophy Company, Nantong, Jiangsu) and drinking water; after feeding for 15 weeks, the mice were euthanized, and intestinal tissue samples were taken for Western Blot and ELISA analysis. The specific analysis methods were as described above.

[0115] 3. Experimental results:

[0116] like Figure 4 b shows that the experimental results show that when feeding TNF △ARE When mice were fed VB6, the shearing of GSDMD downstream of NLRP6 inflammasome in the jejunum and ileum was significantly enhanced compared with mice fed a normal diet, indicating that the activation of NLRP6 inflammasome was significantly enhanced in the absence of VB6. Similarly, the release of inflammatory cytokine interleukin 18 (IL-18) in the intestinal tissue and serum of the VB6-deficient group was significantly higher than that of the normal diet group ( Figure 4 c-4d). The above results all indicate that when vitamin B6 is deficient, TNF ΔARE The activation of NLRP6 inflammasome in intestinal inflammation mice was significantly enhanced, and its downstream GSDMD shearing and IL-18 release were significantly enhanced, indicating that the inflammation in the mice was more severe.

[0117] Results and Discussion:

[0118] The experimental results of Example 4 show that when vitamin B6 is deficient, TNF ΔARE The activation of NLRP6 inflammasome in mice with enteritis was significantly enhanced, and its downstream GSDMD shearing and IL-18 release were significantly enhanced, indicating that enteritis in mice was more severe. ΔARE The mouse model is very similar to human Crohn's disease (CD). We further used the DSS colitis model, which is more similar to human ulcerative colitis (UC), to verify the above conclusions.

[0119] Example 5 Vitamin B6 deficiency significantly aggravates DSS colitis in normal mice

[0120] 1. Experimental model:

[0121] Dextran sulphate sodium (DSS) is a polyanionic derivative of dextran, formed by the esterification reaction of dextran and chlorosulfonic acid, with a molecular formula of (C6H7Na3O14S3)n, a molecular weight ranging from 36000 to 50000, and a sulfur content of 17%-20%. Since the first report of using DSS to prepare a hamster ulcerative colitis model in 1985, a large number of studies have shown that the DSS colitis model is very similar to human ulcerative colitis (UC), and the modeling results show that the experimental animals lose weight, have diarrhea and bloody stools, thicken the intestinal wall, increase the number of infiltrating lymphocytes in the crypts, and have severe intestinal tissue damage. Although its induction mechanism has not yet been clarified, current research mainly believes that DSS increases intestinal permeability, destroys the intestinal mucosal barrier, upregulates certain cytokines (tumor necrosis factor, interleukin, interferon, IL-10 and IL-12), activates certain pathways (NF-κB pathway and TRPV1 pathway) or intestinal flora imbalance. The modeling conditions and operation methods of this model are simple, the cost is low, the reproducibility is good, and it is easy to master and promote. Therefore, this enteritis model is the most widely used.

[0122] 2. Experimental process:

[0123] Normal mice at 5 weeks of age (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into two groups. The first group was given vitamin B6-deficient food (customized vitamin B6-deficient feed, purchased from Jiangsu Nantong Trophy Company) and drinking water; the second group was given normal control food (AIN93G standard feed, purchased from Jiangsu Nantong Trophy Company) and drinking water. After feeding for 3 weeks, 2.5% DSS was added to the drinking water, and the weight and diarrhea of ​​the mice were monitored every day. The disease activity index scoring index is shown in Table 3. When the body weight dropped to about 90% (the fourth day), DSS was removed and normal drinking water was given. When the weight of the mice dropped to about 70% (the eighth day), the mice were sacrificed and samples were taken for analysis.

[0124] Table 3. Disease Activity Index Scores

[0125] score Percentage of weight loss Stool consistency Fecal occult blood 0 0 normal Negative 1 1-5％ Soft stools Light blue 2 5-10％ Mucoid stool blue 3 10-20％ Thin liquid stool Dark Blue 4 ＞20％ Thin liquid stool Naked bloody stool

[0126] 3. Sample collection and processing methods:

[0127] After the mice were killed by cervical dislocation, the complete colon tissue was removed for photographing and length statistics.

[0128] 4. Experimental results:

[0129] Figure 5 Shown is the effect of vitamin B6 deficiency on DSS-mediated colitis in normal mice. Figure 5a shows the process of induction and treatment of DSS-mediated enteritis model in normal mice. Figure 5 b The weight changes of the mouse DSS colitis model showed that from the 7th day, the weight of VB6-deficient mice began to decrease significantly compared with normal mice (****p<0.0001). The degree of diarrhea in mice was significantly higher than that in the control group ( Figure 7 c, **p<0.01, ***p<0.001, ****p<0.0001), the survival rate was significantly reduced, and the length of the colon of the mice was also shorter (Note: when the mouse colon is inflamed, its length will be shortened). It proves that vitamin B6 deficiency significantly aggravates DSS-mediated colitis in normal mice.

[0130] 5. Discussion of results:

[0131] The experimental results of Example 5 showed that vitamin B6 deficiency significantly aggravated DSS-mediated colitis in normal mice, verifying that vitamin B6 can improve inflammatory bowel disease by inhibiting the activation of the NLRP6 inflammasome pathway in vivo. In order to further confirm that VB6 exerts an anti-inflammatory effect through the NLRP6 inflammasome pathway, we will verify the above phenotype in NLRP6 knockout mice.

[0132] Example 6 Vitamin B6 deficiency has no significant effect on inflammatory bowel disease in NLRP6 knockout mice

[0133] 1. Experimental process:

[0134] The 5-week-old NLRP6 knockout mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into two groups. The first group was given vitamin B6-deficient food (customized vitamin B6-deficient feed, purchased from Jiangsu Nantong Trophy Company) and drinking water; the second group was given normal control food (AIN93G standard feed, purchased from Jiangsu Nantong Trophy Company) and drinking water. After 3 weeks of feeding, 2.5% DSS was added to the drinking water, and the weight and diarrhea of ​​the mice were monitored every day. When the body weight dropped to about 90% (the fourth day), DSS was removed and normal drinking water was given. When the weight of the mice dropped to about 70% (the eighth day), the mice were sacrificed and samples were taken for analysis.

[0135] 2. Experimental results:

[0136] Figure 6 Shown is the effect of vitamin B6 deficiency on DSS-mediated colitis in NLRP6 knockout mice. Figure 6 a shows the process of induction and treatment of DSS-mediated enteritis model in NLRP6 knockout mice. Figure 6b As shown in the weight changes of the mouse DSS colitis model, in NLRP6 knockout mice, there was no significant difference in the symptoms of colitis between the VB6 deficiency group and the control group. Figure 6 b), diarrhea score ( Figure 6 c), colon length ( Figure 6 de) and other aspects. This shows that vitamin B6 deficiency has no significant effect on inflammatory bowel disease in NLRP6 knockout mice, further proving that vitamin B6 works by inhibiting the NLRP6 pathway. Next, we will verify whether VB6 addition has a therapeutic effect on inflammatory bowel disease.

[0137] Example 7 Oral administration of vitamin B6 can effectively improve inflammatory bowel disease in mice

[0138] 1. Experimental process:

[0139] Normal mice aged 8 weeks (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into a control group and a VB6-added group. They were given normal food and drinking water supplemented with 2.5% DSS from day 0, and the weight and diarrhea of ​​the mice were monitored every day. When the body weight dropped to about 90% (day 5), DSS was withdrawn, and the control group was given a normal diet and normal drinking water, and the VB6-added group was given a normal diet and drinking water supplemented with 0.6mg / ml VB6. When the body weight of the mice dropped to about 70% (day 10), the mice were sacrificed and samples were taken for analysis.

[0140] 2. Sample collection and processing methods:

[0141] After the mice were killed by cervical dislocation, the complete colon tissue was taken out for photographing and length counting. The colon contents were then rinsed clean and coiled for pathological sectioning and staining.

[0142] 3. Pathological section staining:

[0143] The mouse colon was fixed in formaldehyde for pathological staining and sectioning. First, the sections were placed in xylene I for 10 min-xylene II for 10 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-95% alcohol for 5 min-90% alcohol for 5 min-80% alcohol for 5 min-70% alcohol for 5 min-distilled water for washing; the sections were stained with Harris hematoxylin for 3-8 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water; the sections were stained with eosin stain for 1-3 min; the sections were placed in 95% alcohol I for 5 min-95% alcohol II for 5 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-xylene I for 5 min-xylene II for 5 min to dehydrate and make them transparent, the sections were taken out of xylene and dried slightly, and sealed with neutral gum; microscopic examination, image acquisition and analysis.

[0144] 4. Experimental results:

[0145] Figure 7 The results show that oral administration of vitamin B6 improves inflammatory bowel disease in mice. Figure 7 b The weight changes of the mouse DSS colitis model showed that from the 9th day, the weight of mice fed with vitamin B6 began to increase significantly compared with mice fed with a normal diet (**p<0.01, ***p<0.001). The degree of diarrhea in mice was significantly lower than that in the control group ( Figure 7 c, *p<0.05, **p<0.01, ***p<0.001), and the length of the colon of the mice was also longer (Note: When the mouse colon is inflamed, its length will be shortened). Through H&E (staining of cell nuclei and cytoplasm) pathological staining analysis, it was found that compared with mice on a normal diet, the intestinal wall of the mice in the VB6 supplementation group did not thicken, the number of lymphocytes infiltrating the intestinal crypts was significantly reduced, and the tissue damage score was lower ( Figure 7 f-7g, ****p<0.0001). These results demonstrate that oral supplementation of VB6 can significantly improve the intestinal inflammation phenotype induced by DSS in mice.

[0146] 5. Discussion of results:

[0147] The experimental results of Example 7 showed that oral vitamin B6 can effectively improve inflammatory bowel disease in mice, confirming that VB6 inhibits the release of intestinal inflammatory cytokines by inhibiting excessive activation of NLRP6 inflammasomes in vitro, thereby improving the phenotype of inflammatory bowel disease. Considering that VB6 is often supplemented with intravenous infusion in clinical practice, we tried to verify the effect of VB6 in vivo by intravenous injection, which is a more clinical method and dosage.

[0148] Example 8 Intravenous injection of vitamin B6 can effectively improve inflammatory bowel disease in mice

[0149] 1. Experimental process:

[0150] Normal mice aged 8 weeks (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into a control group and a VB6-added group. They were given normal food and drinking water with 2.5% DSS from day 0, and the weight and diarrhea of ​​the mice were monitored every day. When the body weight dropped to about 90% (day 5), DSS was withdrawn, and the VB6-added group was given normal diet and normal drinking water, and 0.5 mg VB6 was injected into the eye vein every other day. The control group was given normal diet and normal drinking water, and an equal volume of PBS was injected into the eye vein every other day. When the weight of the mice dropped to about 70% (day 11), the mice were sacrificed and samples were taken for analysis.

[0151] 2. Experimental results:

[0152] Figure 8 The figure shows the improvement of inflammatory bowel disease in mice by intravenous injection of vitamin B6. Figure 8 b The weight changes of the mouse DSS colitis model showed that from the 9th day, the weight of mice fed with vitamin B6 began to increase significantly compared with mice fed with a normal diet (*p<0.05, ****p<0.0001). The degree of diarrhea in mice was significantly lower than that in the control group ( Figure 8 c, *p<0.05, **p<0.01, ***p<0.001), the length of the colon of mice was also longer (Note: When the colon of mice is inflamed, its length will be shortened) ( Figure 8 d-8e, *p<0.05). H&E (staining of nuclei and cytoplasm) pathological staining analysis showed that compared with mice fed a normal diet, the intestinal wall of mice in the VB6 supplementation group did not thicken, the number of lymphocytes infiltrating the intestinal crypts was significantly reduced, and the tissue damage score was lower ( Figure 8 f-8g, ****p<0.0001). These results demonstrate that intravenous injection of VB6 supplementation can significantly improve the intestinal inflammation phenotype induced by DSS in mice.

[0153] 3. Results and Discussion

[0154] The experimental results of Example 8 showed that intravenous supplementation of vitamin B6 can also effectively improve inflammatory bowel inflammation in mice, confirming that in in vitro experiments, VB6 inhibits the release of intestinal inflammatory cytokines by inhibiting the over-activation of NLRP6 inflammasomes, thereby improving the phenotype of inflammatory bowel disease.

[0155] Example 9 There is a significant difference in the activation of NLRP6 inflammasomes between healthy people and patients with inflammatory bowel disease

[0156] The activation process of NLRP6 inflammasome is as follows: ① NLRP6 aggregation: NLRP6 undergoes oligomerization after stimulation and interacts with the PYD of ASC through its PYD (pyrin domain). ② ASC polymerization: After ASC is recruited by NLRP6, it interacts with the PYD of NLRP6 through its PYD and further oligomerizes to form a macromolecular complex, commonly known as ASC specks. ③ Caspase-1 activation: Aggregated ASC interacts with the CARD of Caspase-1 through its CARD (caspase recruitment domain), recruiting and activating Caspase-1. ④ Caspase-1 self-cleavage: After Caspase-1 is activated, it undergoes self-cleavage, separating the active p20 and p10 subunits to form activated Caspase-1. ⑤ Maturation of IL-18 and IL-1β: Activated Caspase-1 cleaves the precursor forms of IL-18 (pro-IL-18) and IL-1β (pro-IL-1β), turning them into mature IL-18 and IL-1β and promoting their secretion. (Li R, Zhu S. 2020. NLRP6inflammasome[J]. Molecular Aspects of Medicine, 76: 100859.)

[0157] To confirm that IL-18 secreted in the intestine is mediated by NLRP6 inflammasome, we detected the shearing of GSDMD and the secretion of mature IL-18 in the intestine of NLRP6 inflammasome knockout mice (N6 KO), NLRP3 inflammasome knockout mice (N3 KO), and NLRP9 inflammasome knockout mice (N9 KO). Fig. 9 It can be seen that the loss of NLRP3 and NLRP9 does not affect the secretion of IL-18, but the loss of NLRP6 significantly reduces the secretion of IL-18. Comprehensively considering the activation process of NLRP6, it can be seen that the shearing of GSDMD and the secretion of IL-18 can accurately reflect the activation of NLRP6 inflammasome.

[0158] "NLRP6 inflammasome overactivation" means that under physiological or pathological conditions, the activation of NLRP6 inflammasome exceeds the normal range, leading to excessive inflammatory response and possible tissue damage. In order to more conveniently judge the activation of NLRP6, we can do so by detecting the secretion of IL-18.

[0159] The inventors randomly selected 60 healthy human serum samples from Anhui Provincial Stereoscopic Examination Center and 40 IBD patient serum samples from Shanghai Changhai Hospital, and used the human IL-18 detection kit (Beijing Sino Biological Technology Co., Ltd., catalog number KIT10119) to detect the IL-18 content in each serum sample according to the kit instructions. Fig.10 As shown, there were significant differences in the IL-18 content, i.e., NLRP6 inflammasome activation, between healthy people (HC) and patients with inflammatory bowel disease (IBD / UC / CD) (P < 0.001, P < 0.01). Specifically, the average IL-18 level of healthy people (HC) was 2649.9pg / mL and the median was 1905.7pg / mL, while the IL-18 level of some IBD patients was abnormally elevated, with an average of 5386.6pg / mL and a median of 4188.4pg / mL. After distinguishing between ulcerative colitis (UC) and Crohn's disease (CD) patients in IBD patients, it was found that NLRP6 was overactivated in both UC and CD patients, with an average IL-18 level of 5612.4pg / mL and a median of 4072.7pg / mL in UC patients and an average IL-18 level of 5124.4pg / mL and a median of 3742.6pg / mL in CD patients. Based on this, we further verified whether there was a difference in vitamin B6 treatment in IBD patients with overactivated NLRP6.

[0160] Example 10 Clinical study on the treatment of patients with inflammatory bowel disease by oral administration of vitamin B6

[0161] The clinical treatment study of oral vitamin B6 for patients with inflammatory bowel disease was conducted in Shanghai Changhai Hospital. The clinical trial followed the Declaration of Helsinki and relevant Chinese clinical trial research norms and regulations.

[0162] The inclusion criteria for the subjects were: 1) age: 18-75 years old; 2) patients diagnosed with IBD according to the consensus opinion on the diagnosis and treatment of inflammatory bowel disease (2018, Beijing); 3) complete medical history information; and, 4) voluntary participation in this clinical trial and signing of the informed consent.

[0163] The exclusion criteria for subjects were:

[0164] 1) Patients with peripheral neuropathy; 2) Abdominal abscess, toxic megacolon, fulminant colitis, partial colectomy or total colectomy; 3) Suffering from other autoimmune diseases, blood system diseases, tumors, acute infections, severe liver and kidney dysfunction, serious diseases such as severe anemia, neutropenia, heart failure, organic heart disease, hepatitis B, cirrhosis, kidney disease and mental illness; 4) Taking vitamin B6 tablets in the past 3 months; 5) History of abuse of psychoactive substances; 6) Pregnant or lactating women, or planning to become pregnant in the next 6 months; 7) Neurological diseases: such as Alzheimer's disease, stroke, Parkinson's disease; 8) Participating in other clinical trials in the past 6 months; 9) Incomplete medical records (including gender, age, diagnosis information, colorectal endoscopy results, pathological diagnosis results and other demographic data); or, 10) Currently taking: levodopa, phenobarbital, phenytoin sodium.

[0165] The termination / withdrawal criteria for the study are: 1) the subject actively requests to withdraw from treatment; 2) the subject has an unexpected pregnancy; 3) an intolerable adverse event; 4) the subject does not participate in the follow-up; 5) the researcher believes that the subject is not suitable to continue to participate in the study; or, 6) death.

[0166] The study used a double-blind randomized controlled trial, and randomly assigned participants to the experimental group or the control group according to a computer-generated random number table. Finally, 72 subjects in the experimental group and 72 subjects in the control group were enrolled.

[0167] Experimental group: Based on the standard treatment regimen, vitamin B6 tablets (Hangzhou Minsheng, 10 mg / tablet) were given, 1 tablet each time, orally once a day, for a course of 3 weeks; patients with parenteral nutrition or fasting were given vitamin B6 injection 2g + 0.9% sodium chloride 500ML, intravenous drip, once a day, and after returning to a normal diet, they were changed to oral vitamin B6 tablets, 1 tablet each time, orally once a day, for a total course of 3 weeks. Drug storage method: Store at room temperature.

[0168] Control group: Based on the standard treatment regimen, the patients were given the same type of placebo tablets, taken orally once a day, 1 tablet each time, for a course of 3 weeks; patients with parenteral nutrition or fasting were given 0.9% sodium chloride 500mL, intravenous drip, once a day, and after returning to a normal diet, they were given the same type of placebo tablets for oral treatment, 1 tablet each time, once a day, for a total course of 3 weeks. Drug storage method: Store at room temperature.

[0169] Conventional treatment plans for IBD refer to: Inflammatory Bowel Disease Group of the Chinese Society of Gastroenterology. Consensus on the diagnosis and treatment of inflammatory bowel disease (2018, Beijing)[J]. Chinese Journal of Practical Internal Medicine, 2018, 38(9): 796-813. DOI: 10.19538 / j.nk2018090106.

[0170] Clinical research program on oral vitamin B6 for patients with inflammatory bowel disease Fig.11 As shown, it includes the baseline period (V1) and the treatment period (V2-V5). The details are as follows:

[0171] Oral vitamin B6 tablets (or placebo tablets of the same type) were taken from V1 for 3 weeks. Evaluations were performed at 1, 2, 3, and 6 months after VB6 administration. The observation indicators were as follows: the improvement of the clinical remission rate (Mayo score / simplified CDAI score ≤ 2 points and no single sub-item score > 1 point) of IBD patients after oral vitamin B6 tablets were taken during conventional treatment, serum PLP and IL-18 levels of patients at V1 baseline, and Mayo score / simplified CDAI score at V1 and V5 stages.

[0172] IBD disease activity scoring details:

[0173] Table 4 Simplified Crohn's Disease Activity Index (CDAI) calculation method

[0174]

[0175]

[0176] Note: “-” means no such item. 1) Concomitant diseases include arthralgia, iritis, erythema nodosum, pyoderma gangrenosum, aphthous ulcer, fissure, new fistula and abscess, etc. ≤4 points for remission, 5-7 points for mild activity, 8-16 points for moderate activity, >16 points for severe activity

[0177] Table 5: Modified Mayo Scoring System for Assessing the Activity of Ulcerative Colitis

[0178]

[0179] Note: a Each subject served as his or her own control to evaluate the abnormality of the frequency of bowel movements; b Daily bleeding score represents the most severe bleeding in 1 day; c Physician's overall evaluation includes 3 criteria: subject's review of abdominal discomfort, overall well-being and other manifestations, such as physical examination findings and subject's performance status; score ≤ 2 points and no single sub-item score > 1 point indicates clinical remission; 3-5 points indicate mild activity; 6-10 points indicate moderate activity; 11-12 points indicate severe activity.

[0180] By collecting the changes in Mayo scores, simplified CDAI scores, and clinical remission rates of all 12 IBD patients in the trial group who had completed five follow-ups (other patients were still in the group undergoing clinical trials) in stages V1 and V5 after oral vitamin B6 administration, and combining mass spectrometry to detect serum PLP levels in patients at baseline in V1, and enzyme-linked immunosorbent assay (Human IL-18 ELISA Kit, Invitrogen, catalog number KHC0181) to detect serum IL-18 levels in patients at baseline in V1, we found that there were significant differences in serum IL-18 levels in IBD patients before treatment (e.g. Fig.12 As shown). Further analysis showed that the serum IL-18 level before treatment was positively correlated with the difference between the Mayo score and the simplified CDAI score in the V1 and V5 stages, that is, patients with higher IL-18 levels before treatment had more significant disease improvement (Mayo / CDAI score difference) after VB6 treatment, that is, vitamin B6 is more suitable for treating patients with inflammatory bowel disease with over-activation of NLRP6. When the patient is an inflammatory bowel disease patient without over-activation of NLRP6 (IL-18 is lower than 1500pg / mL), it indicates that the use of vitamin B6 treatment is ineffective. This is basically consistent with the results in Example 9, and the slight differences in the specific data may be due to different detection kits.

[0181] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Application of vitamin B6 in the preparation of NLRP6 inhibitors.

2. Use of vitamin B6 in the preparation of drugs for preventing and / or treating diseases caused by NLRP6 inflammasome.

3. The use according to any one of claims 1-2, characterized in that: The vitamin B6 is a combination of one or more of pyridoxine, pyridoxal, pyridoxamine, pyridoxine phosphate, pyridoxal phosphate or pyridoxamine phosphate.

4. The use according to claim 3, characterized in that: The vitamin B6 is preferably pyridoxal phosphate.

5. According to the use according to any one of claims 1-4, vitamin B6 can bind to the PYD domain of NLRP6.

6. The use according to claim 2, characterized in that: The medicine also includes pharmaceutically acceptable carriers and adjuvants.

7. Use of the drug according to claim 2 in combination with one or more of the following in the preparation of a drug for NLRP6 inflammasome-induced diseases; (1) agents that increase the efficacy of vitamin B6 on IL-18 inhibitors; (2) agents that increase the inhibitory efficacy of vitamin B6 on NLRP6; (3) Agents that enhance the ability of vitamin B6 to target the PYD domain of NLRP6.

8. Use of NLRP6 inhibitors in the preparation of drugs for preventing and / or treating diseases caused by overexpression of TNFα.

9. A method for preparing an animal model of NLRP6 overactivation, the method comprising feeding an animal with a vitamin B6-deficient food for at least 21 days, thereby obtaining an animal model of NLRP6 overactivation.

10. Use of the NLRP6 over-activated animal model obtained by the preparation method according to claim 9 in screening drugs for preventing and / or treating diseases caused by NLRP6 inflammasome.

Images