| Background and objective:The etiology and pathogenesis of the Inflammatory bowel disease(IBD) is still unclear, but the abnomal immunne response result from the over-activation of the Toll-like receptor(TLR) signaling pathway has play an important role in this disease, however, the specific mechanism of the over-activation of the TLR is still unknown. SIGIRR is a newly found negative regulator of the TLR signaling pathway. It can reduce inflammation and put the breaks on Toll-like receptors, but the specific mechanism is still unknown. Some researches had demonstrate that SIGIRR is low expressed in IBD, but there is no report about whether the SIGIRR plays an important part in the pathogenesis of IBD and it can inhibit the inflammation result from the over-activation of TLR. Chitosan nanoparticle was a kind of non-toxic material of gene transfer vectors. Therefore, this research will focus on the relations between SIGIRR and the changes of the TLR signaling pathway in mice model of experimental colitis. We will use the newly gene delivery vector of chitosan nanoparticles loaded exogenous gene encoding SIGIRR to administer through gut, and observe its effect on the TLR signaling pathway of the experimental colitis mice model. So we will clarify whether the SIGIRR has the treatment of IBD and the possible mechanism for the treatment of IBD, and provide a new target and direction for the treatment of IBD.Method:1. Prepration of the Chitosan/pUNO-mSIGIRR and chitosan/pUNO nanoparticles:①Use the extraction kit to extract the pUNO-mSIGIRR and pUNO plasmid;②Use the compound coacervation method to prepare chitosan-nanoparticles, we will optimize the following five factors which will affect the prepration of the chitosan-nanoparticles: concentration of the chitosan, pH, plasmid concentration, Na2SO4 concentration, vortexing time; ③Use the method of transmission electron microscopy to observe the morphology of nanoparticles; Use photon correlation spectroscopy to determine the size of the nanoparticles; Use the fluorescence photometric spectrometer to analyze the free DNA in the suspension and calculate the encapsulating efficiency.2. Groups: Seven mice in each group, enemaed 0.1ml each time.①Control group: only given normal sodium;②Model group: DSS-induced experimental colitis mice and given normal sodium;③Empty plasmid group:model mice and given full dose chitosan/pUNO nanoparticles;④Half dose group: model mice and given equal volume of normal soium diluted chitosan/pUNO-mSIGIRR nanoparticles;⑤Full dose group:model mice and given whole amount of chitosan/ pUNO-mSIGIRR nanoparticles;3. Establishment of DSS-induced colitis and intervention of drugsBALB/c mice were given 5% DSS ad libitum for 7 days, Acute colitis model was successfully established according to anyone of the symptoms, loose stools, diarrhea, positive occult blood and bloody stools. Mice only given normal sodium were used as the control group. After that, mice were randomly divided into groups mentioned above and drugs were given by enteroclysis once a day, from the second day of model building to the end. Then mice were sacrificed and samples were collected.4. Observation and detection of each index①To observe the weight and disease activity index (DAI) of each group everyday;②To observe the magnum shape of colon in mice macroscopically, and the change of pathohistology through HE stain;③To detect the expression of SIGIRR mRNA in colon through RT-PCR;④To detect the protein expression of TLR4, MyD88, SIGIRR and NF-κB p65 through immunohistochemistry stain. Result:1. Research about the preparation and the physical and chemical properities of the chitosan nanoparticles:We will optimize the following five factors which will affect the prepration of the chitosan/pUNO and chitosan/pUNO-mSIGIRR nanoparticles: the optimum preparation condition is concentration of chitosan(300ug/ml), concentration of plasmid(100ug/ml), concentration of Na2SO4 (8 mM), vortexing time (30s), pH (5.5).①Use the transmission electron microscope to observe the shape of nanoparticles, and the chitosan nanoparticles were almost spherical and conform to the shape comparison rules, and the mean diameter is less than 100nm.②Use the photon correlation spectroscopy to analyze the ZAve diameter, intensity mean diameter, number mean diameter, volume mean diameter and polydispersity of the chitosan/pUNO and chitosan/pUNO-mSIGIRR nanoparticles. The results of the chitosan/pUNO-mSIGIRR nanoparticles were 213.8nm, 215.8nm, 80.43nm, 104nm and 0.466. The results of the Chitosan/pUNO nanoparticles were 269.6nm, 366.6nm, 67.74nm, 450.4nm and 0.251.③Use the luminosity spectrometer to detemine the free DNA content in the suspension, the encapsulating efficiency is 99.9% .2. The effect of the chitosan/pUNO-mSIGIRR nanoparticles on the experimental mice colitis and its mechanism.(1) Weight and DAI score of mice:①Except the control group, the weights of mice in each group begin to decline and on the 5th day to the lowest, then rose gradually in the following days. The weight descending percentages of model group and drug groups were higher than that of control group on the 5th, 10thday(P<0.001, 0.05);The weight descending percentages of half dose group and full dose group were lower than that of model group and empty plasmid group on the 5th, 10thday, but without statistical significance(P>0.05). There is also no significant difference between the half dose group and full dose group(P>0.05).②There were significant deviation of DAI scores of each group on the 5th, 10th day, the DAI scores of control group were significantly lower than those of model group and each drug group(P<0.001); on the 5th day the scores of half dose group and full dose group were significantly lower than those of model group(P<0.05), half dose group and full dose group are lower than the empty plasmid group, but without statistical significance(P>0.05); on the 10th day model group and empty plasmid group are higher than the half dose group and full dose group, but also without statistical significance(P>0.05).(2) Pathological change of colon①Magnum shape of colon mucosa: There was no congestion, edema and erosion in colon mucosa of control group; there was obviously congestion, edema and erosion in colon mucosa of model group and empty plasmid group; the symptoms of congestion, edema and erosion of the half dose group and full dose group were obviously lighter than those of model group and empty plasmid group in colon mucosa.②The pathohistological severity score of colon: The degrees of colon inflammation, pathological depth and crypt destruction in control group were significantly lower than those in other groups(P<0.001, 0.005, 0.05); the degrees of those indexes in half dose group and full dose group were significantly lower than those in model group and empty plasmid group (P<0.01, 0.05).(3) SIGIRR expression in colon mucosaThe expression of SIGIRR mRNA in colon mucosa of the control group was significantly higher than those of model group and empty plasmid group(P<0.05); half dose group and full dose group were significantly higher than model group and empty plasmid group(P<0.001, 0.05); half dose group and full dose group were higher than control group, but without statistical significance(P>0.05); full dose group were higher than half dose group, but without statistical significance(P>0.05); The protein expression of SIGIRR in the control group was significantly higher than those of model group and empty plasmid group(P<0.001); half dose group and full dose group were significantly higher than those of model group and empty plasmid group(P<0.001); Nevertheless,there is no significanct difference between half dose group and full dose group(P>0.05).(4)TLR4 expression in colon mucosaThe protein expression of TLR4 in glandular cell: the control group was significantly lower than those in model group and empty plasmid group(P<0.001),half dose group and full dose group were significantly lower than model group(P<0.05), the full dose group were significantly lower than empty plasmid group(P<0.05). The protein expression of TLR4 in inflammatory cell: the control group was significantly lower than those in model group and all drug groups(P<0.001, 0.01), half dose group and full dose group were significantly lower than model group(P<0.01) and empty plasmid group(P<0.05).(5)MyD88 expression in colon mucosaThe protein expression of MyD88 in glandular cell and inflammatory cell: the control group was significantly lower than those of model group and all drug groups (P<0.001, 0.05), half dose group and full dose group were significantly lower than model group and empty plasmid group(P<0.001, 0.05).(6)NF-κB p65 expression in colon mucosaThe protein expression of NF-κB p65 in inflammatory cell: The control group was significantly lower than those of model group and all drug groups (P<0.001, 0.05), half dose group and full dose group were significantly lower than model group and empty plasmid group(P<0.001, 0.05),but there is no statistical significance between half dose group and full dose group(P>0.05).Conclusion:1. In this experiment, We use the compound coacervation method to prepare the chitosan/pUNO-mSIGIRR nanoparticles, its ZAve diameter, intensity mean diameter, number mean diameter, volume mean diameter and polydispersity and encapsulating efficiency is consistent with the requirements of gene therapy vectors.2. Use the compound coacervation method to prepare chitosan nanoparticles and administered them to the mice through the gut, the expression of the SIGIRR mRNA and protein of the drug groups were higher than the model and empty plasmid groups.This demonstrate that SIGIRR can enter cells for expression. These suggest that chitosan nanoparticles can be used as gene delivery vectors for gene therapy.3. The expression of SIGIRR was decreased in the colon mucosa of DSS- induced experimental colitis mice, while the expressions of TLR4, MyD88, NF-κB p65 increased, which indicated that the over-activation of TLRs/NF-κB signaling pathway and the decrease of its negative regulatory mechanism had involved the pathogenesis of IBD.4. Chitosan/pUNO-mSIGIRR nanoparticles can ease the symptoms of colon and attenuate the inflammation and injury of pathohistology, which can be taken as a new approach in the treatment of IBD.5. The expressions of TLR4, MyD88, NF-κB p65 significantly decreased in colon mucosa of mice which were given chitosan/pUNO-mSIGIRR nanoparticles, and were concerned with the attenuation of colon inflammation, suggesting that SIGIRR may attenuate the tissue inflammation through the inhibition of TLRs/NF-κB activation. |
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