| Background and objecyive:Sinomenine is a pure alkaloid extracted from the Chinese medical plantsinomenium acutum. It was demonstrated that sinomenine had a wide range ofpharmacological actions, including anti-inflammatory, antirheumatic, analgesic,antiarrhythmic, and immunosuppressive effects in the previous studies. Nowsinomenine has been a common prescription drug used for rheumatoid arthritis inchina. Recent studied have shown that sinomenine inhibits lymphocytes proliferation,macrophage immigration and pro-inflammatory cytokine production. Sinomenine canalso modify imbalance of ratio of T cell subtypes and increase apoptosis ratio ofspleen lymphocyte. The recently researches confirmed that Inflammatory boweldisease (IBD) is similar with symptom of the immunized disease that dysimmunity isinduced by lymphocytes in pathogenic process. Although the etiology andpathogenesis of IBD is still unclear, but the abnormal immune response result fromthe over-activation of the Toll-like receptor (TLRs) signaling pathway has play animportant role in this disease. Given that the anti-inflammatory, antirheumatic andimmunosuppressive effects of sinomenine, we hypothesize that sinomenine improvescolonic inflammation and is preventive from the over-activation of TLRs and thedeseasing SIGIRR expression in IBD. The aim of this study, therefore, is to evaluatethe effect of sinomenine that is loaded within the oral colon specific drug deliverysystem on DSS-induced colitis in mice and its possible mechanism.Methods：1. Preparation and in vitro release of eudragit coated chitosan microspherescontaining sinomenine for colon targeting.Use the aldehyde cross-linking method to prepare chitosan microspheres, wewill utilize orthogonal experiment to optimize the following four factors which willaffect the prepration of the chitosan microspheres: concentration of the chitosan,cross-linking agent usage, the phase volume ratio of oil and aqueous, vortexing velocity. Use the method of scanning electron microscopy and light microscope toobserve the morphology of microspheres and determine the size of the microspheres;Use the ultraviolet spectrophotometry to analyze content and calculate theencapsulating efficiency, drug-loading rate and release rate. Furthermore, the releaserate of microspheres coated the differently rate acrylic resin will be determined in thedifferent medium solutions so as to evaluate the microspheres slow-release and targetin mice colon.2. Groups: Six mice in each group, enemaed0.2ml each time. The groups weredissolved in0.5%carboxymethyl cellulose sodium salt solution as the solvent fordrug excipient suspending, the sinomenine microsphere containing a dose equivalentto the administration of sinomenine groups.①Normal control group (N): unmodeling mice only administrated normal0.5%CMC solutions;②Model group (M): DSS-induced experimental colitis mice and administrated0.5%CMC solution;③Salicylazosulfapyridine group (SASP): model mice and administrated100mg/kg SASP0.5%CMC solution;④Chitosan microsphere group (C): model mice and administrated540mg/kgchitosan microspheres0.5%CMC solution;⑤Low dose sinomenine group (LSN): model mice and administrated30mg/kgsinomenine0.5%CMC solution;⑥Middle dose sinomenine group (MSN): model mice and administrated90mg/kg sinomenine0.5%CMC solution;⑦High dose sinomenine group (HSN): model mice and administrated270mg/kgsinomenine0.5%CMC solution;⑧Low dose sinomenine microsphere group (LSNM): model mice andadministrated180mg/kg sinomenine chitosan microspheres (contained30mgsinomenine)0.5%CMC solution;⑨Middle dose sinomenine microsphere group (MSNM): model mice andadministrated540mg/kg sinomenine chitosan microspheres (contained90mgsinomenine)0.5%CMC solution; ⑩High dose sinomenine microsphere group (HSNM): model mice andadministrated1.6g/kg sinomenine chitosan microspheres (contained270mgsinomenine)0.5%CMC solution;3. Establishment of DSS-induced colitis and intervention of drugsBALB/c mice were given5%DSS ad libitum for7days, Acute colitis modelwas successfully established according to the symptoms, loose stools, diarrhea,positive occult blood and bloody stools. Mice only given normal sodium andenemaed with0.5%CMC solution were used as the control group. After that, micewere randomly divided into groups mentioned above and drugs were given byenteroclysis once a day, from the second day of model building to the tenth day 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 groupeveryday;②To observe the magnum shape of colon in mice macroscopically, and thechange of pathohistology through HE stain;③To detect the expression of TLR4, MyD88and SIGIRR mRNA in colonthrough RT-PCR;④To detect the protein expression of TLR4, MyD88and SIGIRR throughWestern blotting;⑤To detect the protein expression of MyD88and NF-κB p65throughimmunohistochemistry stain.Results：1. The orthogonal designed preparation condition and in vitro release characterresearch of sinomenine chitosan microspheres:We use the orthogonal design to optimize the following four factors which will effectthe preparation of the sinomenine chitosan microspheres: the optimum preparationcondition is the concentration of chitosan15mg/ml, the phase volume ratio of oil andaqueous1:10, the volume of cross-linking agent3ml and stirring velocity600rpm4h,temperature40℃. Use the scanning electron to observe the shape of microspheres,and the chitosan microspheres were almost spherical and few conglutination within each other. The mean diameter is range between10um and60um to15.61±7.50um.Use the ultraviolet spectrophotometry to analyze content and calculate theencapsulating efficiency is59.00±13.26%and drug-loading rate is19.10±4.51%. Themean diameter that using acrylic resin Ⅱ,Ⅲ coated microspheres were determinedby light microscope reach to20.72±8.03μm, the encapsulating efficiency is67.67±1.20%. Without release of sinomenine from the microspheres were observed inthe artificial gastric juice (PH1.2) and in the artificial intestinal juice (PH4.5),respectively. However, the release of sinomenine was startly increased in artificialintestinal juice (PH6.8), in the artificial large intestinal juice (PH7.4) and containingrat colon contents, which was near to7%,60%and70%at5h,24h and24h,respectively. According to the optimal formulation prepared sinomemine chitosanmicrospheres have good shape and encapsulation efficiency, and meet therequirements of colon-spcific drug delivery.2. The therapeutic effects of colon-specific Sinomenine loaded chitosanmicrospheres in mice model with experimental colitis and its mechanisms.(1) Colon length, Weight and DAI score of mice:①In the normal control group mice colon length were9.88±0.50cm. The modelgroup and chitosan microsphere group were7.15±1.38cm and7.18±0.45cmrespectively; significantly shorter than the normal control group (P＜0.05). Exceptthat the low dose sinomenine group were7.48±0.89cm no discrepancy with the modelgroup. The SASP group, middle and high dose sinomenine group, low, middle andhigh sinomenine microsphere group were8.67±0.75cm,8.83±1.00cm,9.30±0.56cm,9.51±0.66cm,9.73±0.83cm,9.75±1.28cm, respectively; its more significant deviationthan the model group. Except the normal control group, the weights of mice in eachgroup begin to decline and on the4thday to the lowest, then rose gradually in thefollowing treatment days. The weight descending percentages of model group andchitosan microsphere group were higher than that of control group, SASP group,sinomenine groups and microspheres groups on the experimental day(P<0.001,0.05)；The weight descending percentages of SASP group and sinomenine groups andsinomenine microspheres were lower than that of normal group but without statisticalsignificance(P>0.05). There is also no significant difference between the SASP and sinomenine each dose groups and sinomenine microsphere each dose groups(P>0.05).②There were significant deviation of DAI scores of each group on the5th,10thday, the DAI scores of normal control group were significantly lower than those ofmodel group and each drug group (P<0.001); on the5thday the scores of normalgroup, model group, SASP group, chitosan group, sinomenine each dose groups andsinomenine microsphers each dose groups were0,8.67±1.51,3.17±0.41,9.17±0.41,1.50±0.55,0.83±1.17,0.67±1.21,1.17±1.17,0.17±0.41,0.50±0.41, respectively.Each drugs groups were significantly lower than those of model group and chitosangroup (P<0.001). There are no statistics deviation between sinomenine middle highgroups and sinomenine microsphere each group. On the10thday model group andchitosan group are higher than drugs each groups, but its also without statisticalsignificance (P>0.05), the DAI score of drugs group were all decrease and conform totime dependency.(2) Pathological change of colon①The shape of mice colon mucosa: There was no congestion, edema anderosion and ulcer in colon mucosa of normal control group; there was obviouslycongestion, edema, gassiness and erosion in colon mucosa of model group andchitosan microsphere group; the symptoms of congestion, edema and erosion of theSASP group and sinomenine middle, high groups and sinomenine microsphere eachgroups were obviously lighter than those of model group and chitosan microspheregroup in colon mucosa.②The pathohistological severity score of colon: The degrees of coloninflammation, pathological depth and crypt destruction in different doses ofsinomenine groups were significantly differences at the score (F=35.69, P<0.001;F=27.98, P<0.001;F=10.79, P<0.001), normal group were significantly lower than themodel group and drug groups (P<0.001,0.005,0.05); SASP group and sinomeninemiddle and high dose groups were significantly lower than the model group andsinomenine low dose group (P <0.01,0.05), between SASP, sinomenine middle andhigh dose groups no significant difference. In the same dosage from different doses ofsinomenine microspheres groups were significantly difference at histopathology score (F=74.57, P=0.00; F=72.81, P=0.00; F=54.29, P=0.00). The model group andchitosan group was significantly higher than normal group and sinomeninemicrosphere all doses groups (P <0.001,0.05), between SASP group and sinomeninemicrosphere middle and high dose groups were no significant difference (P>0.05).Sinomenine groups were significantly higher than that of sinomenine microspheregroups corresponding to the same doses groups (P <0.001,0.005,0.05).(3)TLR4expression in mice colon tissue①The mRNA expression of TLR4in different doses of sinomenine groups weresignificantly differences at the score (F=100.40, P<0.001), between the two groupsfound that the model group was significantly higher than the normal group and drugsgroups (P <0.01), except the sinomenine low dose group, between the sinomeninemiddle and high dose groups and SASP no significant difference (P>0.05). In thesame dosage from different doses of sinomenine microspheres groups weresignificantly difference at the score (F=157.50, P<0.001). The model group andchitosan group was significantly higher than that in the normal group and sinomeninemicrosphere all doses groups (P <0.01). SASP group drugs was significantly higherthan sinomenine microspheres middle and high dose groups (P <0.01), and the thesinomenine microspheres dose groups were dose-dependent decrease. Sinomeninegroups were significantly higher than that of sinomenine microsphere groupscorresponding to the same doses groups (P <0.01).②The protein expression of TLR4in different doses of sinomenine groups weresignificantly differences at the score (F=64.13, P<0.001), between the two groupsfound that the model group was significantly higher than the normal group and drugsgroups (P <0.01), sinomenine all dose groups that were dose-dependent decline weresignificantly higher than the SASP group (P <0.01), the low dose group wassignificantly higher than the high dose group (P <0.01). In the same dosage fromdifferent doses of sinomenine microspheres groups were significantly difference atthe score (F=118.38, P<0.001). The model group and chitosan group was significantlyhigher than that in the normal group and sinomenine microsphere all doses groups (P<0.01). SASP group was significantly lower than sinomenine microspheres low dosegroup.(P <0.01), and between SASP and the the sinomenine microspheres middle and high dose groups were no statistical significance (P>0.05). Sinomenine groupswere significantly higher than that of sinomenine microsphere groups correspondingto the same doses groups (P <0.01).(4)MyD88expression in colon mucosa①The mRNA expression of MyD88in different doses of sinomenine groupswere significantly differences at the score (F=18.26, P<0.001), between the twogroups found that the model group was significantly higher than the normal groupand drugs groups (P <0.01), and there was no significant difference between themodel group and the low dose sinomenine group. SASP group and sinomenine highdose group have no significant difference (P>0.05), the sinomenine all dose groupsshowed dose-dependent decrease. In the same dosage from different doses ofsinomenine microspheres groups were significantly difference at the score (F=29.02,P<0.001). The model group and chitosan group was significantly higher than that inthe normal group and sinomenine microsphere all doses groups (P <0.01). Nosignificant different between the SASP group and sinomenine microsphere all dosegroups (P>0.05), and sinomenine microsphere groups showed dose-dependentdecrease. The sinomenine low and middle groups were significantly higher than thesame dose sinomenine microsphere groups (P <0.05), and the sinomenine high dosegroup was higher than the sinomenine micrpsphere high dose group, but no statisticaldifferentce (t=1.06, P=0.313).②The protein expression of MyD88in different doses of sinomenine groupswere significantly differences at the score (F=19.33, P<0.001), between the twogroups found that the model group was significantly higher than the normal groupand drugs groups (P <0.01). Between SASP group and the sinomenine middle andhigh dose groups have no significant difference (P>0.05), the sinomenine all dosegroups showed dose-dependent decrease. In the same dosage from different doses ofsinomenine microspheres groups were significantly difference at the score (F=17.21,P<0.001). The model group and chitosan group was significantly higher than that inthe normal group and sinomenine microsphere all doses groups (P <0.01). SASPgroup and sinomenine microsphere all dose groups have no significantly difference(P>0.05), and the sinomenine microsphere all dose groups showed dose-dependent decline. The sinomenine each dose groups was significantly higher than the samedose of sinomenine microsphere groups.③The protein expression of MyD88in mice colon mucosa of inflammatory cellsin different doses of sinomenine groups were significantly differences at the score (H=22.642, P<0.001), between the two groups found that the model group wassignificantly higher than the normal group and drugs groups (P <0.01). SASP groupand the sinomenine middle and high dose groups have no significant difference (P>0.05). In the same dosage from different doses of sinomenine microspheres groupswere significantly difference at the score (H=28.606, P<0.001). The model group andchitosan group was significantly higher than that in the normal group and sinomeninemicrosphere all doses groups (P <0.01). SASP group and sinomenine microsphere alldose groups have no significantly difference (P>0.05), and the sinomeninemicrosphere all dose groups showed dose-dependent decline. The sinomenine eachdose groups was higher than the same dose of sinomenine microsphere groups, butwithout statistical significance (P>0.05).(5) SIGIRR expression in colon mucosa①The expression of SIGIRR mRNA in colon mucosa in different doses ofsinomenine groups were significantly differences at the score (F=34.20, P<0.001),between the two groups found that the model group was significantly lower than thenormal group and drugs groups (P <0.01), SASP group and sinomenine all dosegroups have no significant difference (P>0.05). In the same dosage from differentdoses of sinomenine microspheres groups were significantly difference at the score(F=45.53, P<0.001). The model group and chitosan group was significantly lowerthan that in the normal group and sinomenine microsphere all doses groups (P <0.01).No significant different between the SASP group and sinomenine microsphere alldose groups (P>0.05), and sinomenine microsphere groups showed dose-dependentincrease. The sinomenine low and middle groups were lower than the same dosesinomenine microsphere groups, but no statistically significant (t=-2.07, P=0.066; t=0.24, P=0.817), and the sinomenine high dose group was significant higher thanthe sinomenine micrpsphere high dose group (t=-4.34, P=0.001).②The expression of SIGIRR protein in colon mucosa in different doses of sinomenine groups were significantly differences at the score (F=22.30, P<0.001),between the two groups found that the model group was significantly lower than thenormal group and drugs groups (P <0.01), the SASP group significantly higher thanthe sinomenine low dose group (P <0.01), but no significant difference with thesinomenine middle and high dose groups (P>0.05), the sinomenine all dose groupsshowed dose-dependent increase. In the same dosage from different doses ofsinomenine microspheres groups were significantly difference at the score (F=16.98,P<0.001). The model group and chitosan group was significantly lower than that inthe normal group and sinomenine microsphere all doses groups (P <0.01). Nosignificant different between the SASP group and sinomenine microsphere all dosegroups (P>0.05). The sinomenine low group was significantly lower than the samedose sinomenine microsphere groups,(t=-2.07, P=0.020), and the sinomeninemiddle and high dose groups were higher than the sinomenine micrpsphere middleand high dose group but no statistically significant (t=-0.91, P=0.386; t=0.64,P=0.536).(6)NF-κB p65expression in colon mucosaThe protein expression of NF-κB p65in mice colon mucosa of inflammatorycells in different doses of sinomenine groups were significantly differences at thescore (H=31.016, P<0.001), between the two groups found that the model group wassignificantly higher than the normal group and drugs groups (P <0.01). The SASPgroup and the high dose groups have no significant difference (P>0.05), andsignificantly lower than the sinomenine low and middle dose groups (P <0.01). In thesame dosage from different doses of sinomenine microspheres groups weresignificantly difference at the score (H=35.351, P<0.001). The model group andchitosan group was significantly higher than that in the normal group and sinomeninemicrosphere all doses groups (P <0.01). SASP group and sinomenine microsphere alldose groups have no significantly difference (P>0.05), and the sinomeninemicrosphere all dose groups showed dose-dependent decline. The sinomenine eachdose groups was significantly higher than the same dose of sinomenine microspheregroups (P<0.05). Conclusion：1. In this experiment, we use the orthogonal optimization experiments screeningthe sinomenine chitosan microsphere preparation conditions of the prepared chitosanmicrospheres that size distribution, encapsulation efficiency, drug loading and releaserate comply with the requirements of the colon targeted release formulations.2. The expression of TLR4, MyD88and NF-κB p65were increased in the colonmucosa of DSS-induced experimental colitis mice, while the expression of SIGIRRdecreased,that indicidated the over-activation of TLRs/NF-κB signaling pathway andthe decrease of its negative regulatory mechanisum participate in the pathopoiesis ofIBD.3. The sinomenine and the sinomenine chitosan microspheres could relieve thesymptoms of inflammatory and pathological damage in experimental colitis mice.TLR4, MyD88, and NF-κB p65expression was significantly decreased, and SIGIRRexpression in a dose-dependent increase in the mice colone tissue. The Sinomeninemay be through the inhibition of TLRs/NF-κB activation and the negative regulationof SIGIRR to reduce the inflammation of the colon tissue.4. The same dose of sinomenine microspheres in mice with experimental colitissymptoms and impact of the TLR signaling pathway is superior to the sinomeninegroup, suggesting that the dose can be reduced through the drug-loaded microspheresto improve drug treatment in the colon concentration. The role of the sustainedrelease and targeted can be expected to become the new ways of Chinese medicinetreatment of IBD. |
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