Chalcone-derivative L6H21 attenuates the OVA-induced asthma by targeting MD2

Abstract

Asthma represents a significant global challenge that affects individuals across all age groups and imposes substantial social and economic burden. Due to heterogeneity of the disease, not all patients obtain benefit with current treatments. The objective of this study was to explore the impact of MD2 on the progression of asthma using L6H21, a novel MD2 inhibitor, to identify potential targets and drug candidates for asthma treatment. To establish an asthma-related murine model and evaluate the effects of L6H21, ovalbumin (OVA) was used to sensitize and challenge mice. Pathological changes were examined with various staining techniques, such as H&E staining, glycogen staining, and Masson staining. Inflammatory cell infiltration and excessive cytokine secretion were evaluated by analyzing BALF cell count, RT-PCR, and ELISA. The TLR4/MD2 complex formation, as well as the activation of the MAPK and NF-кB pathways, was examined using western blot and co-IP. Treatment with L6H21 demonstrated alleviation of increased airway resistance, lung tissue injury, inflammatory cell infiltration and excessive cytokine secretion triggered by OVA. In addition, it also ameliorated mucus production and collagen deposition. In the L6H21 treatment group, inhibition of MAPK and NF-кB activation was observed, along with the disruption of TLR4/MD2 complex formation, in contrast to the model group. Thus, L6H21 effectively reduced the formation of the MD2 and TLR4 complex induced by OVA in a dose-dependent manner. This reduction resulted in the attenuation of MAPKs/NF-κB activation, enhanced suppression of inflammatory factor secretion, reduced excessive recruitment of inflammatory cells, and ultimately mitigated airway damage. MD2 emerges as a crucial target for asthma treatment, and L6H21, as an MD2 inhibitor, shows promise as a potential drug candidate for the treatment of asthma.

Keywords: Asthma; Chalcone derivative; MD2 inhibitor; MD2 recombination; TLR4.

Fig. 1

MD2 expression in asthmatic patients is elevated than those in non-asthmatic patients. immunohistochemistry for MD2 (200X) A Non-asthmatic patient A, B non-asthmatic patient B, C asthmatic patient A, D asthmatic patient B

Fig. 2

L6H21 relieving OVA-triggered AHR in mice. A Chemical structure of L6H21. B Experimental schemes of mouse asthma model. OVA/Al(OH)₃ was adopted to sensitized mice on day 0 and day 14; from day 25 to day 31, 1% OVA aerosol was used to expose the mice for 7 continuous days. C Airway resistance in mice. According to the evaluation, airway responsiveness indicated the mean response of mice with mechanical ventilation to risen Mch doses (mean ± SEM; n = 6 in the respective group; **P < 0.01 in comparison with the OVA group)

Fig. 3

L6H21 attenuated OVA-triggered histopathologic changes and inflammatory cell recruitment. A H&E staining(200X) was performed to identify inflammatory cell infiltration and tissue damage of the lung in mice. B Histological analysis was conducted on lung tissues to calculate lung inflammatory score. C, D After the OVA challenge was completed, L6H21 decreased eosinophils (D) and total cells (C) within BALF (results have an expression of the mean ± SEM; n = 6 in the respective group; *P < 0.05, **P < 0.01 in comparison with the OVA group)

Fig. 4

L6H21 attenuated OVA-triggered IgE, Th2 cytokines, and inflammatory factor secretion. A L6H21 ameliorate OVA-triggered increase in IgE in serum. B, C Effects of L6H21 on attenuating OVA-triggered increase in IL-4 (B), IL-5 (C) and, IL-13 (D) in BALF. E, F RT-qPCR was performed to examine TNF-α (F) and IL-6 (E) mRNA level in lung tissue. G, H ELISA was adopted to examine TNF-α (H) and IL-6 (G) protein level in BALF (results have an expression of the mean ± SEM; n = 6 in the respective group; *P < 0.05, **P < 0.01 in comparison with the OVA group)

Fig. 5

L6H21 attenuated OVA-triggered goblet cell hyperplasia and airway remodeling. A Goblet cell hyperplasia in the airway epithelium was identified using PAS staining (200X). B Masson staining (200X) was performed to identify collagen deposition of the airway in mice. Goblet cell scores C were evaluated by histological analysis of lung tissues. D, E Relative mRNA level of TGF-β (D) and Col IV (E) were examined through RT-qPCR (results have an expression of the mean ± SEM; n = 6 in the respective group; *P < 0.05, **P < 0.01 in comparison with the OVA group)

Fig. 6

L6H21 attenuated OVA-triggered activation of the MAPK, NF-кB pathways, and combination of TLR4 and MD2 in mouse lung tissue. A Part of the lung tissue was collected and homogenated to detect the phosphorylation of P38, ERK, and JNK and the decomposition of IкB by Western blotting assay. B–E Relative density of P-JNK/JNK (B), P-ERK/ERK (C), P-P38/P38 (D), and IкB/GAPDH (E) were obtained. F Part of the lung tissue was collected and homogenated to detect the combination of TLR4 and MD2 by co-immunoprecipitation. G Relative density of TLR4/MD2 were obtained (results have an expression of the mean ± SEM; n = 4 in the respective group; *P < 0.05, **P < 0.01, ***P < 0.001, in comparison with the OVA group)

Fig. 7

Mechanism diagram of Chalcone derivative L6H21 attenuates the OVA-induced asthma by targeting MD2. [By Figdraw (www.figdraw.com)]