Formononetin Inhibits Allergic Diseases By Regulating E-Cadherin Through TLRs

With the continuous development of society, the cases of allergic diseases gradually increased in China. The cytokines TSLP and IL-33, which are secreted by ECs under external stimuli, are the key factors. Studies had shown that the TSLP mRNA expression in airway ECs and Keratinocytes could be significantly increased by TLR (2、3、4、8、9) ligands. At the same time other studies showed that the reducing expression of E-Cadherin in epithelial cells could also lead to the increase of TSLP expression. But, what’s the connection between TLRs, E-Cadherin and initiative key factors of allergic diseases (TSLP and IL-33)? Could TLRs influence the expression of TSLP and IL-33 by regulating the E-Cadherin? This is the problem which should be solved in this paper. Yupingfengsan administrated in the recurrence phase can reduce the recurrence of allergic diseases. But the substantial basis and mechanism of effects of Yupingfengsan remains not clear. Our previous study found that there was high level of Formononetin in the serum containing Yupingfengsan. By the establishment of Th2-type allergic dermatitis model, we found administrating Formononetin in the initial stage can significantly relieve the symptoms of inflammation in mice, suggesting that Formononetin had an effect on allergic diseases and it affected in the initial stage. But how did it play a role in the early stage of allergic disease? And what is the mechanism of its anti-allergic effect? Based on the preliminary findings, this research article will carry out from the following aspects:1 The regulation of E-Cadherin by TLRs on epithelial cells and its possible role in allergic diseases(1) We research whether TLRs could regulate the E-Cadherin between epithelial cells, and whether this regulation will affect the initiative key factors (TSLP and IL-33) derived from epithelial cells. By administrating TLR (2、3、4、8、9) ligands to 16HBE cells for 24h, we observe the expression and distribution of E-Cadherin between epithelial cells, and detect protein and gene expression levels of TSLP and IL-33 derived in epithelial cells.(2) After this, we will find which TLRs have effects on the distribution and expression of E-Cadherin between epithelial cells, and then, we can study its mechanism of effects further. TLR3 and TLR4 ligands can activate a variety of signal transduction pathways, including PI3K and MAPK. But it remains unclear by which signal path they affect the distribution of E-cadherin. So we added ERK1/2 inhibitors, p38MAPK inhibitors, JNK1/2/3 inhibitors and PI3K inhibitors, and observed the expression of E-cadheirn in epithelial cells stimulated with TLR4 and TLR3.2. Mechanism research on Formononetin inhibiting allergic diseases(1) By using mouse allergic contact dermatitis (Allergic Contact Dermatitis, ACD) model and relapse model, study the role of Formononetin on the intervention in allergic inflammation and preventing allergic inflammation recrudescence. BALB/c mice were sensitized by FITC to become the ACD model, and the mouse was administrated with Formononetin continuously until Day3. At Day7, we use FITC attacking the ears and measure the thickness after 24h, calculate ear swelling (thickness of the right ear-left ear thickness) and compare the weight changes of the left and right ears. Ears were also examined for pathologic changes. In ACD recurrence model, we administrated Formononetin in remission stage for 10 days, and attack right ears of mice at 48 hours after the last administration. Then we measured mice’s ears to calculate ear swelling. Ears were examined for pathologic changes. We also detected the levels of IL-4, IL-5, IL-9 and IL-13 in the right ear tissue homogenate.(2) We examine the effect of Formononetin on TSLP and IL-33 derived from epithelial cells. Establish the Th2-type ACD model in initial stage, and give Formononetin in early stage. Then, detect protein and gene levels of TSLP and IL-33 in ear tissues. Stimulate the bronchial epithelial cells 16HBE with Poly (I:C) and LPS in vitro, and observe the effect of Formononetin on expression of TSLP and IL-33.(3) We observed the effect of Formononetin on expression and distribution of E-Cadherin between epithelial cells, to reveal whether the effect of Formononetin on expression and distribution of E-Cadherin. Establish the Th2-type ACD model and the ACD recurrent model, and give Formononetin in initial stage or remission stage. Then we observe the connection between epithelial cells with electron microscopy and detect the distribution and expression of E-Cadherin between epithelial cells in mice ear tissues. In vitro, we stimulate bronchial epithelial cells 16HBE with TLR ligands, and observe the regulation of Formononetin on the distribution and expression of E-Cadherin between epithelial cells.(4)To reveal that whether the Formononetin regulated the distribution and expression of E-Cadherin between epithelial cells by regulating TLRs and their downstream signaling molecules in ACD model, we observed the effect of Formononetin on TLRs-NF-κB major signaling molecule in the Th2-type ACD model, the expression of TLR(3-.4) protein in ECs and downstream signal molecules. We established the Th2-type ACD model in initial stage and given Formononetin in early stage, then we detected the expression of TLRs-NF-κB major signaling molecules in ears, to investigate the effects of Formononetin on TLRs-NF-κBmajor signaling molecules. In vitro, stimulate epithelial cells with Poly (I:C) and LPS. Then, we detected TLR3 and TLR4 protein levels and expression of downstream signaling molecules.Research results:(1) In this study, we found that TLR3 and TLR4 agonists could be contribute to the disorder of the distribution of E-cadherin between epithelial cells. Moreover, only TLR3 agonists can down regulate the expression of E-cadherin. The TSLP and IL-33 expression was significantly increased in epithelial cells which were stimulated by LPS and Poly(I:C), indicating that LPS and Poly (I:C) can promote the secretion of TSLP and IL-33 by activating TLR4 and TLR3 to disrupt the E-cadherin between epithelial cells.(2)We find that after Poly(I:C) stimulated, adding p38MAPK inhibitors 20uM, PI3K inhibitor 20μM, ERK1/2 inhibitor 20μM and JNK1/2/3 inhibitor 20μM to epithelial cells can increase the expression of E-Cadherin, where p38MAPK inhibitors and PI3K inhibitor are the most evident. It suggests that the TLR3 regulation on E-Cadherin between epithelial cells is mainly through p38MAPK and PI3K signal transduction pathway. In addition, we stimulate epithelial cells with LPS, the expression and distribution of E-Cadherin between cells improved significantly after adding four inhibitors. This suggests that TLR4 may regulate E-Cadherin between epithelial cells through p38MAPK, PI3K, ERK1/2 and JNK1/2/3 transduction pathways.(3) The Formononetin can alleviate the ear inflammation on ACD mice. It can reduce swelling of the ears, improve local tissue congestion and inflammatory cell infiltration and other pathological changes. In the relapse ACD model, after administrating Formononetin 10 days in remission and stimulated the mice at 48h after the last dose, Formononetin can inhibit the ear swelling in mice, reduce the thickness and weight of the ears, inflammatory cell infiltration and reducing ear edema. In addition, Formononetin also reduced to the expression levels of IL-4、 IL-5、 IL-13.(4)ACD model in initial stage was established and administrated with Formononetin at the initial stage. We found that there was significantly increase in gene and protein levels of IL-33 and TSLP in the ears of mouse in model group compared to the control group. But the gene and protein levels of IL-33 and TSLP were significantly decreased in Formononetin group. We stimulated the epithelial cells with Poly(I:C) and LPS, and observed the effects of Formononetin on TSLP and IL-33 expression. We found that Formononetin could inhibit TSLP and IL-33 production, which agreed with in vitro results. This more confirmed that the basis of Formononetin administrated at initial stage which could cure allergic diseases was closely related with regulating the expressing of initiative key factors TSLP and IL-33 which secreted by epithelial cells.(5) To reveal whether Formononetin regulating expression of pro-allergic key factors TSLP and IL-33 is related with regulating the expression of E-Cadherin between epithelial cells, we observe the effect of Formononetin on E-Cadherin expression and distribution between epithelial cells. Establishing FITC-induced allergic contact dermatitis (ACD) model and ACD relapse model, we use electron microscopy and find that the gap and connection between the epithelial cells was significantly decreased, E-cadherin expression was reduced and its distribution was without order. Administrating Formononetin in the early sensitization phase, E-cadherin expression was increased and its distribution was much improved. Exoteric experiment results showed that with Poly(I:C) and LPS stimulation, distribution of E-Cadherin proteins between cells became significantly disorder, and the Poly(LC) could make a difference in the amount of E-Cadherin protein between epithelial cells. After administrating Formononetin, not only the distribution of E-Cadherin protein between cells had some improvement, but also the content of E-Cadherin protein was raised.(6) We detected the TLRs-NF-κB primary signaling molecular in the ears of ACD early phase model mice, found that TLR2, TLR3, TLR4, TLR9, MYD88 and NF-KB gene levels in model group rise significantly compared to the control group. After administrating Formononetin, gene expression of TLR3, TLR4, MYD88,TLR9 and NF-κB was inhibited. With Poly(I:C) stimulated in 16HBE cells, the TLR3 downstream molecules ERK1/2, p38MAPK, JNK and PI3K phosphorylation were increased significantly compared to the control group. After administrating Formononetin, the phosphorylation of TLR3 downstream signaling molecules PI3K was significantly reduced, The TLR4 downstream signaling molecules ERK1/2 and p38MAPK was also significantly reduced. This suggests that Formononetin may improve the expression and distribution of E-Cadherin between the epithelial cells by regulating TLR3 and TLR4 to affect the activation of their downstream signaling molecule PI3K(TLR3), ERK1/2 and p38MAPK(TLR4).Conclusion:(1)We can conclude that TLR3 influences the expression and distribution of epithelial cells E-Cadheirn protein by p38MAPK and PI3K signal path, and then promotes key pro-allergic cytokine TSLP and IL-33 secreting, thereby promoting Th2-type allergic diseases. But TLR4 makes an effect on epithelial cells E-Cadheirn protein distributing by p38 MAPK、PI3K、ERK1/2 and JNK1/2/3 signal paths.(2)Formononetin may improve the E-Cadheirn protein expressing and distributing in epithelial cells by adjusting the TLRs (TLR3、4), then suppress the Th2-type allergic diseases and reduce recurrence.