Cimifugin Reduces The Recurrence Of Allergic Inflammation Based On The Regulation Of MicroRNAs On Epithelial Junction Proteins

Allergic disease is the immune response caused by antigenic stimulation with tissue injury or dysfunction. Allergies have the characteristics of rapid onset, intense reaction and fast regression and always recurrent when the organism contact with allergen again. It is now the hot potato for researchers to figure out how to control allergic disease progression, improve physical fitness and reduce recurrence. MicroRNAs (miRNAs) is a kind of endogenous non-coding single small RNA. It has been reported that miRNAs can regulate the balance of Thl/Th2 and play important roles in atopic dermatitis, allergic rhinitis and asthma. But the dynamic changes of miRNAs in the process of allergic disease, especially in the remission of allergic diseases recurrence is unclear. YupingfengSan(YPFS) is a classic formula that derived from "Dan Xi Xin Fa", consists of astragalus, radix sileris and atractylodes and it has been widely used clinically in allergic diseases. Our previous studies have shown that YPFS can significantly inhibit allergic inflammation in mice. YPFS alleviated the inflammatory responses dramatically when it is administrated at the remission of recurrent allergic contact dermatitis (ACD) model. At the meantime, we found a high level of cimifugin in the YPFS-containing serum. Furthermore, cimifugin administrated in effective phase and induction phase of ACD significantly inhibited allergic inflammation. But it is unclear that what is (are) the accurate role (s) of cimifugin at the initial stage and remission of ACD recurrence since it showed significant inhibiting effect.This paper includes the following contents:Firstly, we investigated the pathogenesis of recurrent allergic dermatitis focusing on microRNAs (miRNAs). A FITC-induced allergic contact dermatitis recurrence murine model was built for observing the pathological process of allergic diseases. After the ear swelling recover to a normal level or so (remission), we used FITC to elicit more serious inflammation (recurrence). We measured the changes of miRNAs in remission by gene chips, and then validate the expression of miRNAs in remission by Real time-PCR. Then we observed the dynamic changes of miRNAs in the various stages of the ACD recurrence model. The prediction of specific miRNAs targets were done through miRanda, microcosm and TargetScan. To study the effect of miRNAs on connexin protein in epithelial, we selected the target genes associated with connexin proteins and validated its expression in vitro.Secondly, we investigated the effect of cimifugin in ACD and ACD recurrence model. Cimifugin was administrated to observe the effects on ear swelling, immune organ and histopathology. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IL-4, IL-5, IL-9, and IL-13 at the initial phase of ACD model. In ACD recurrence model, cimifugin was administrated for 10 days in remission. Then we elicited the inflammation again 48 hours after the latest administration. We measured mice’s ears to calculate ear swelling. Ears were collected for pathological changes. We also detected the level of IL-4, IL-5, IL-9 and IL-13 in the supernatant of right ear homogenate.Thirdly, we investigated the effect of cimifugin on epithelial junction proteins in vivo and miR-155-5p/miR-146a-5p in vitro. At the initial phase of ACD model, we performed an electron microscopy observation on the connection between epithelial cells and we measured the expression levels of epithelial junction proteins including CLDND1, CLDN-1 and Occludin to evaluate the effect of cimifugin. All the experiments were also performed at remission and recurrence phase of ACD recurrence model. Furthermore, the effect of cimifugin on the expression of miR-155-5p and miR-146a-5p were explored in vitro. The results showed that: 1. MiRNAs were differently expressed in the remission of ACD recurrence model and miR-155-5p could regulate the epithelial junction proteins.The results of miRNAs microarray showed that there are 55 miRNAs upregulated and 73 downregulated compared with the control group at the remission of ACD recurrence model. We validated differentially expressed genes at the remission of ACD recurrence model including miR-155-5p, miR-146a-5p, miR-3473b, miR-3473d, miR-677-3p, miR-770a-5p and miR-5119. The results showed that miR-146a, miR-155-5p and miR-3473b significantly increase at the remission while miR-770a-5p and miR-5119 are significantly downregulated at the remission.Investigation of miR-155-5p, miR-146a-5p, miR-3473b, miR-770a-5p and miR-5119 in each period of ACD recurrence model (initial phase, elicitation, remission and the secondary elicitation) showed that miR-146a-5p, miR-155-5p and miR-3473b significantly increase at the beginning of sensitization and continue to increase in remission while miR-770a significantly reduce at the elicitation phase and continue to reduce at the remission phase.We predicted the specific miRNAs targets by miRanda, microcosm and TargetScan. We found that CLDN16 and CLDND1 are the targets of miR-155-5p. CLDN16 and CLDND1 are the members of claudin family. In HaCaT cells treated with TNF-a and Poly:(I:C) and other stimulations, the expression of mir-155 significantly increased while the target genes CLDN16 and CLDND1 decreased. Meanwhile, the expression of TJs including Occludin, CLDN-1 and ZO-1 were decreased, suggesting that mir-155-5p targeted CLDN16, CLDND1 and regulated the expression of other junction proteins.2. Cimifugin administrated at the initial stage of ACD or the remission stage of ACD recurrence model could inhibit allergic inflammation.Cimifugin (12.5 mg·kg-1,50 mg·kg-1) could not only alleviate the ear inflammation with ear swelling and inflammatory cell infiltration of ACD mice, but also inhibit the expression of IL-4, IL-5, IL-9 and IL-13 in the supernatant of ear homogenate. Cimifugin administrated in remission for ten consecutive days could inhibit ear swelling, inflammatory cell infiltration and the expression of inflammatory cytokine of ACD recurrence model. The results showed that cimifugin could regulate the immune status of organisms under pathological condition with long-term effect.3. Cimifugin reduced the recurrence of allergic inflammation by affecting the regulation of miR-155-5p on epithelial junction proteins.We investigated the mechanism of cimifugin in ACD and ACD recurrence model. An electron microscopy observation has been performed on FITC-sensitized ears, which showed separated gap among the epithelial cells. Cimifugin alleviated pathologic status and increased the expression of key TJs proteins CLDND1, CLDN-1 and Occludin at the elicitation phase and remission stage in ACD recurrence model. Furthermore, cimifugin inhibited the expression of mir-155-5p in vitro. The results showed that cimifugin alleviated the recurrence of ACD by inhibiting the abnormal expression of miR-155-5p, which could restore CLDND1, CLDN16 and Occludin and reduce the expression of Th2 inflammatory cytokines.Conclusion:1. MiRNAs were differently expressed in the remission of ACD recurrence model. And miR-155-5p could down-regulate the epithelial junction proteins including CLDN16, CLDND1 and Occludin.2. Cimifugin alleviated the recurrence of ACD by inhibiting the abnormal expression of miR-155-5p, which could restore CLDND1, CLDN16 and Occludin and reduce the level of Th2 inflammatory cytokines.3. Intervene in miRNAs of allergic disease remission may be an effective way to reduce allergic disease recurrence.