The Study Of The Absorption Through Skin Of The Flexible Nanonipsomes Of Methotrexate For External Application And The Influence On The Level Of Interferon-gamma(IFN-γ) And Interleukine 4(IL-4) In Serum Of Rats With Rheumatoid Arthritis(RA)

OBJECTIVE:Through researching of the effectiveness of theabsorp- tion through skin of the Flexible Nanonipsomes of Methotrexate(MTX) for external application and the influence on interferon-γ(IFN-γ)and interleukin-4 (IL-4) to find out a new type of treatment rheumatoidarthritis (RA) that convenience and take effect quickly but poisonous sideeffect small.METHODS:1. Penetrate through the skin experiment: Used themethod of Chenying to prepare ex vivo rat skin and installed the skin onthe modified Franz-diffusion cells, the horny layer faced the suppliedroom and the dermis layer faced the absorbed cell. The absorbed liquorwas physiologic saline and the capacity was 30 ml. The temperature waspermanent (37±1)℃. The rotation speed of magnetic stirrer was 100 perminute. Applying the 0.5% concentration of methotrexate, the 0.25%concentration of flexible nanonipsomes methotrexate and the 0.5%concentration of flexible nanonipsomes methotrexate on the surface ofhorny layer separately and uniformly. Then used the 0.45um microporefilm to filter on the 2nd,the 4th,the 8th, the 10th and the 12th hour. The filtratewere attenuated to 10ml by physiologic saline and determine theabsorbance on 306nm. Subtracting the absorbance of the physiologicsaline, we used the absorbance in standard curve to compute theaccumulated transmit dose and the accumulated transmissivity of 12hours.2. Detect the level of IFN-γand IL-4: Among 85 SD rats, 10 wereregarded as normal group, the other 75 rats were modeled by collagenⅡand complete Freund's Adjuant. The CIA model was well-known modelof the RA rats. 5 rats were lost because of the unsuccessful modeling andtotally 70 rats were randomly divided into 4 groups: 10 rats wereregarded as the model group, 20 rats were regarded as the 0.5％ concentration of MTX group, 20rats were regarded as the 0.25％concentration of nanonipsomes MTX group and 20 rats were regarded asthe 0.5％concentration of nanonipsomes MTX group. Rats in normalgroup were drunk water freely, the other groups were administered at the8th day after modeled: rats in model group were painted with distilledwater once a week, the other groups painted with 2.0g to 4.0g (accordingto the size of the articles) the 0.5％concentration of methotrexate, the0.25％concentration of flexible nanonipsomes methotrexate and the 0.5％concentration of flexible nanonipsomes methotrexate discerned on theright postpedes once a week. To evaluate the symptoms of arthritis withthe integral of arthropathy index. The higher of the index, the moreserious of the arthropathy. We used enzyme linked immunosorbent assay(ELISA) to detect the cytokines level of IFN-γand IL-4 in the serum ofrats on the 45th day.RESULTS: 1. The comparison of the quantity of percutaneousabsorption of each rats group: The accumulated transmit dose and theaccumulated transmissivity of 12 hours of the 0.25％concentration ofnanonipsomes MTX and the 0.5％concentration of nanonipsomes MTXwere higher than the 0.5％concentration of MTX for external applicationand the difference was significance (P＜0.01). The accumulated transmitdose and the accumulated transmissivity of 12 hours were no significantdeviation between the 0.25％concentration of nanonipsomes MTX andthe 0.5％concentration of nanonipsomes MTX (P＞0.05).2. The comparison of the integral of arthropathy index of each ratsgroup: The integral of arthropathy index has no significant deviation inall the groups before the 14th day. The integral of arthropathy index ofmodel group was increased as the time. The integral of arthropathy indexof the 0.5％concentration of MTX group, 0.25％concentration ofnanonipsomes MTX group and the 0.5％concentration of nanonipsomesMTX group were at the peak on the 25th day, but were lower than themodel group (P＜0.01). 3. The comparison of the IFN-γlever of each rat groups: The IFN-γlevel of the model group, the 0.5％concentration of MTX group, 0.25％concentration of nanonipsomes MTX group and the 0.5％concentrationof nanonipsomes MTX group were obviously higher than the normalgroup (P all＜0.01). The IFN-γlevel of the 0.5％concentration of MTXgroup, 0.25％concentration of nanonipsomes MTX group and the 0.5％concentration of nanonipsomes methotrexate group were obviously lowerthan the model group (P all＜0.01). The IFN-y level of the 0.25％concentration of nanonipsomes MTX group and the 0.5％concentrationof nanonipsomes MTX group were obviously lower than the 0.5％concentration of MTX group (P all＜0.01). The IFN-γlevel were nosignificant deviation between the 0.25％concentration of nanonipsomesMTX group and the 0.5％concentration of nanonipsomes MTX group(P＞0.05).4. The comparison of the IL-4 lever of each rat groups: The IL-4level of the model group, the 0.5％concentration of MTX group, 0.25％concentration of nanonipsomes MTX group and the 0.5％concentrationof nanonipsomes MTX group were obviously lower than the normalgroup (P all＜0.01). The Il-4 level of the 0.5％concentration of MTXgroup, 0.25％concentration of nanonipsomes MTX group and the 0.5％concentration of nanonipsomes methotrexate group were obviouslyhigher than the modle groups (P all＜0.01). The IL-4 level of the 0.25％concentration of nanonipsomes MTX group and the 0.5％concentrationof nanonipsomes MTX group were obviously higher than the 0.5％concentration of MTX group (P all＜0.01). The IL-4 level were nosignificant deviation between the 0.25％concentration of nanonipsomesMTX group and the 0.5％concentration of nanonipsomes MTX group(P＞0.05).CONCLUSIONS: 1 The Flexible Nanonipsomes of MTX forexternal application can penetrate through the skin effectively.2 The Flexible Nanonipsomes of MTX for external application can decrease the integral of arthropathy index of the rats with RA.3 The effect of the Flexible Nanonipsomes of MTX decrease theserum IFN-γlevel and increase the IL-4 level is better than the normalexternal application of MTX.