IBD conventional induction therapy is safer immunosuppressant?
- Categories:Stomach healthy
- Time of issue:2020-12-02
IBD conventional induction therapy is safer immunosuppressant?
- Categories:Stomach healthy
- Time of issue:2020-12-02
Calcineurin (CN) is a key enzyme for T cell activation. CN dephosphorylates the nuclear factor (NFAT) transcription factor of activated T cells, thereby inducing its transport to the nucleus, while enhancing the expression of T cell-dependent cytokines and regulatory molecules, such as IL-2, interferon-γ, and tumor necrosis Factor-α. Immunosuppressive therapy for IBD (inflammatory bowel disease) can strongly inhibit CN's phosphatase activity. Commonly used drugs include cyclosporine A and tacrolimus, but these drugs can cause serious adverse reactions, including bone marrow suppression and malignancy Increased risk of transformation, teratogenesis, and oligospermia. CysA and FK506 are alternatives to immunosuppressive therapy for IBD. However, these drugs can also cause adverse reactions such as nephrotoxicity, neurotoxicity, fibrosis and hypertension. Due to the limitations of drugs, the long-term application in many chronic inflammations or autoimmune diseases is limited. Therefore, it is very necessary to develop safer immunosuppressants.
Polaprezinc (Polaprezinc, PZ) is a chelate composed of zinc and L-carnosine, which is clinically used to treat gastric ulcers. Studies have shown that polyprezinc can treat ulcerative colitis. However, there are few studies on the effect of polyprezinc on the immune system, especially the activity of CN in the colonic mucosa. The role of CN in the colonic mucosa and its inhibitory effect remain to be clarified. This study examined the therapeutic effect of polyprezinc on experimentally-induced colitis rats by inhibiting CN.
1 Materials and methods
1.1 Cells and cell culture
Jurkat T cells (human T lymphocyte line) were cultured in 37°C (5% CO2) RPMI1640 medium, and the medium was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin/glutamine. 1.2 Determination of CN activity The protein sample or purified CN (0.1μM) and calmodulin (0.2μM) were mixed in 200μl reaction mixture (100 mM Hepes-NaOH, pH 7.5, 1 mM CaCl2, 5 mM MgCl2, 0.2 mM NiSO4, 3 mM) pNPP, and different concentrations of Zn²+ or PZ) incubate for 30 minutes. The activity in the presence of EGTA was measured in the same reaction mixture, and 0.2 mM NiSO4 was used instead of 5 mM EGTA. The reaction was stopped by adding 50μl 1M Na2CO3. Read the absorbance value at 405nm with a spectrophotometer. CN activity is calculated as the difference between total phosphatase activity and the activity in the presence of EGTA. 1.3 NFAT activated T cells were pre-incubated with 1μM ionomycin and 0.1μM FK506 or polyprezinc for 30min. The cells were lysed with lysis buffer, and Western blot analysis was performed with monoclonal anti-NFAT1. NFAT activation was determined by protein band shift. 1.4 IL-2 mRNA expression Use high-purity RNA isolation kit to extract total RNA. Using 2μg total RNA as a template, RT-PCR was performed with AccessQuick RT-PCR system under thermal cycling conditions: denaturation at 94°C for 5 min, denaturation at 95°C for 30 cycles for 45s, primer annealing at 60°C for 45s, primer extension at 72°C for 2 min, 72°C The final extension is 10 minutes. 1.5 Determination of cell growth and viability The WST-8 cell proliferation reagent colorimetric method was used to determine cell growth. After adding WST-8 cell proliferation reagent, incubate for 3h. The number of viable cells is estimated by measuring the absorbance at 450nm in the microplate reader, and the reference wavelength is 650nm. All experiments were repeated 6 times. After 72 hours of incubation with PZ, the cell viability was determined by trypan blue staining. Cell viability refers to the number of viable cells (unstained)/total cell number (stained and unstained).
2 Experimental colitis in rats
7-week-old male Wistar rats weighing about 220-270 g were selected. Fasting for 24 hours before inducing colitis, and drinking freely.
2.2 Induction of colitis
Insert a plastic tube with an outer diameter of 2 mm into the anus, with the tip extending 8 cm from the anal edge. To induce colitis, 30 mg of 2,4,6 trinitrobenzene sulfonic acid (TNBS) dissolved in 0.5 ml of 50% ethanol was injected into the lumen. After the administration of TNBS, the animals maintain a head-down posture for about 60 seconds to prevent leakage of the infusion.
2.3 Polyprezinc treatment
Rats were randomly divided into 3 groups (n=6 in each group): normal control group, TNBS control group, and PZ treatment group. PZ was diluted in 1ml 0.5% sodium carboxymethyl cellulose at a dose of 60mg/kg/day. From 24h after TNBS action to the end of the experiment, it was administered rectal once a day. Both the normal control group and the TNBS control group received intracavitary administration as a sham treatment.
2.4 Colon injury assessment
Rats were sacrificed on the 7th day of treatment. Take the 8cm part of the distal colon, rinse it with saline, cut it open, and weigh it. According to the following criteria, the mucosal damage was measured and macroscopically scored. The scoring criteria are: 0, no injury; 1, hyperemia, no ulcer; 2, hyperemia and thickening of the intestinal wall, no ulcer; 3, one ulcer, no thickening of the intestinal wall; 4, two or more ulcers and Inflammation; 5. Two or more major parts of ulcers and inflammations or one part of ulcers and inflammations extends >1cm along the length of the colon; from 6 to 10, at the first 2 cm beyond the first 2 cm, an increase of 1 cm is added . 2.5 Determination of CN activity in colonic mucosa
Use DIAX 600 homogenizer to homogenize mucosal scrapings (about 100 mg) in 0.5 ml of buffer solution, collect the supernatant after centrifugation at 20000×g for 15 minutes, and then use 25 μg of protein sample taken from colonic mucosa according to the above method Determination of CN activity.
2.6 Protein expression detection
Western blot analysis of protein samples taken from colon mucosa with anti-NFAT1 and anti-calcineurin Aα antibodies. The western blot was displayed with the ECL-Plus system and subjected to density analysis.
2.7 Determination of inflammatory cytokine mRNA in rat colonic mucosa
Total RNA is prepared from frozen tissue by a high-purity RNA tissue kit. Using 1μg total RNA as a template, the forward and reverse primers of rat IL-1β, IL-2 and IL-2 were studied respectively.
2.8 Statistical analysis
The results are expressed as mean ± standard deviation. Paired or unpaired t test and analysis of variance were used for statistical analysis, and then the Tukey-Kramer or Dunnett multiple comparison test was performed. p<0.05 is statistically significant. Statistical analysis was performed using StatFlex v6.0
3 Results and analysis
3.1 Polyprezinc inhibits CN activity
"The effect of polyprezinc and ZnSO4 on CN activity. Both polyprezinc and ZnSO4 significantly inhibit CN activity in a dose-dependent manner.
3.2 Polyprezinc inhibits NFAT dephosphorylation of T cells
In order to study the effect of PZ on the activity of CN in vivo, the effect of different concentrations of polyprezinc on the dephosphorization of CN physiological substrate NFAT1 was analyzed by Western blot. In T cells, NFAT1 protein showed rapid migration in SDS-PAGE after treatment with ionomycin. The rapid dephosphorylation of protein ATCN is a sign of its phosphorylation. This rapid migration was completely inhibited by the CN inhibitor FK506. After polyprezinc pretreatment, the rapid migration was reduced in a dose-dependent manner.
3.3 Polyprezinc inhibits IL-2 gene expression in T cells
"IL-2 expression is induced through T cell receptor stimulation with the mitogen Con A, followed by activation of CN and mitogen-activated protein kinase pathways in T cells. To study the effects of polyprezinc, ZnSO4 and FK506 on the expression of IL-2 mRNA in T cells. The expression of IL-2 gene was not detected without stimulation. The expression of IL-2 gene can be induced by Con A stimulation, and the expression can be enhanced by the additional stimulation of TPA and ionomycin. Both polyprezinc and ZnSO4 inhibited IL-2 gene expression in a dose-dependent manner. FK506 completely inhibits IL-2 gene expression.
3.4 Polyprezinc inhibits T cell growth without affecting cell viability
In order to study the effect of polyprezinc on the growth of T cells by inhibiting CN, T cells were cultured for 96 h in the presence or absence of different concentrations of polyprezinc. Although the cells grew without polyprezinc, their growth was significantly inhibited in a dose-dependent manner in the presence of polyprezinc. Cell viability is not affected by PZ. In order to study the effect of polyprezinc on CN's inhibitory effect on the growth of T cells, T cells were cultured with polyprezinc for 96 hours, and the cells grew normally without polyprezinc, but when the cells contained polyprezinc Growth was inhibited in a dose-dependent manner. Cell viability is not affected by polyprezinc.
3.5 The effect of polyprezinc on TNBS-induced colitis in rats
After administration of TNBS in the colon, rats developed colitis, accompanied by symptoms of diarrhea, collapse, erection, anorexia and weight loss. TNBS can induce severe inflammation and ulcers in the distal colon of rats within 3-5 days, and polyprezinc treatment significantly improves these changes in the colon. Polyprezinc treatment significantly reduces the wet weight and inflammation of the colon, indicating that the symptoms of colitis have been significantly improved.
3.6 Activation of CN in colonic mucosa and inhibitory effect of Polyprezinc on it
In order to study the mechanism of polyprezinc in the colon, the activity of CN phosphatase in colon tissue was measured. Compared with the normal control group, CN phosphatase activity was significantly increased in TNBS-induced colitis, which was significantly inhibited after treatment with polyprezinc. CN activity in colon tissue is significantly correlated with colon injury score. In vitro experiments were also carried out. After polyprezinc pretreatment, the CN activity of the mucosa was significantly inhibited in a dose-dependent manner.
3.7 Increased expression of CN protein in colonic mucosa and activation of NFAT and the inhibitory effect of PZ on it
In order to clarify the mechanism of the increase of CN activity in colitis, the expression of CN protein was measured by Western blot analysis, and it was found that the expression of CN protein in TNBS-induced colitis increased compared with the normal control group. Polyprezinc pretreatment inhibits the increase of CN expression. Next, to investigate whether the activation of NFAT1, the dephosphorylation of CN and the nuclear translocation of proteins are affected by colitis induction and polyprezinc. TNBS exposure promoted these changes and increased the protein expression of NFAT1. However, polyprezinc pretreatment reduced these changes.
3.8 Increased expression of inflammatory cytokine genes in colonic mucosa and the inhibitory effect of polyprezinc
In order to further understand the molecular mechanism of polyprezine inhibiting colitis, RT-PCR method was used to detect the expression level of key cytokine mRNA in colonic mucosa that is involved in the regulation of inflammation. As shown in Figure 8, the mRNA of pro-inflammatory cytokines (IL-1β, IL-2, IL-4, IL-6, IFN-γ, TNF-α, MIP-2 and CINC) in TNBS-induced colitis The level is significantly increased in TNBS-induced colitis. However, polyprezine attenuated these increases.
Research shows that CN is activated in the mucosa of experimental colitis in rats. Polyprezinc treatment can significantly inhibit the activation of CN and inhibit the expression of pro-inflammatory cytokines in the mucosa, thereby improving ulcerative colitis. In in vitro experiments, polyprezinc inhibited CN activity, NFAT activation, interleukin-2 expression and T cell growth. And studies have confirmed that polyprezinc does not affect cell viability at effective concentrations. Relaisheng-Polyprezinc Granules inhibit the activation of CN in the rat colonic mucosa and inhibit the gene expression of pro-inflammatory cytokines, thereby improving ulcerative colitis. Compared with traditional immunosuppressive agents that can cause serious adverse reactions, Polyprezinc does not produce any serious adverse reactions due to immune protein complexes. The results of this study provide a theoretical basis for the clinical application of polyprezinc as an immunosuppressive agent in patients with IBD. In addition to promoting mucosal healing, it is another target worth exploring for the treatment of IBD.
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