Study On The Mechanism And Effects Of Polyphenol-rich Blue Honeysuckle Extract On Silica-induced Lung Injury In Mice Via Nrf2/HO-1 And MAPK Signaling

Blue honeysuckle(Lonicera caerulea L.)is belonging to Caprifoliaceae and honeysuckle,is rich in polyphenols,and has anti-inflammatory,anti-oxidant,anti-viral,hypolipidemic,hypoglycemic and immuneenhancing effects.Previous studies have found that polyphenol-rich blue honeysuckle extract(BHE)has protective effects on heart,liver and kidney.However,there is few researches focus on the effect of BHE on lung disease.Long term pulmonary exposure to silica leads to silicosis that manifests progressive interstitial fibrosis.It is characterized by chronic lung inflammation and irreversible fibrosis.There is no effective treatment for silicosis,therefore,the study on the mechanism and effects of BHE on silica-induced lung inflammation and fibrosis,which provides theoretical references for seeking a potential drug or health care product for preventing the pathogenesis of silicosis,is of great significance.In this study,we established an experimental silicosis mouse model and an in vitro silicosis model to explore the immune mechanism and inhibiting effect of BHE on silica-induced lung inflammation and fibrosis,according to inflammatory cytokines,apoptosis-related proteins,oxidative stress,antioxidant capacity,MAPK and Nrf2/HO-1 signaling.The main findings are as follows:Acidic ethanol solution was used to extract blue peony polyphenols according to ultrasonic assisted method.Based on single factor experiment,the extraction process was optimized by response surface analysis.According to the simulation experiment,the optimal extraction process of BHE was: material-liquid ratio 1:25(g/m L),extraction temperature 40 °C,ethanol volume fraction 55%.Under the conditions of the experiment,the content of BHE was 8.52 ± 0.02 mg/g.We used macroporous resin purification method to analyze the adsorption and desorption effects of six macroporous resins on BHE.The results showed that D101 macroporous resin had the best adsorption and desorption effect on BHE.According to the single factor experiment,the most suitable dynamic adsorption and desorption conditions for macroporous resins: adsorption equilibrium time was 12 h,desorption equilibrium time was 2 h,sample concentration was 2.6 mg/m L,ethanol concentration was 95%.The optimal conditions for dynamic adsorption and desorption of BHE: sample velocity was 4 BV/h,sample volume was 520 m L,and elution velocity was 5 BV/h.Under these conditions,the crude extract of BHE was purified by D101 macroporous resin,and the purity of polyphenol was increased from 10.32% to 72.87%.Analysis of the components in the extract using HPLC-ESI-MS2 and HPLC,we found that each milligram of BHE was filled with 0.41 mg of cyanidin-3-glucoside and 0.22 mg of chlorogenic acid,which was the main polyphenols component in the BHE.An in vitro model of silicosis was established using the MH-S cell line,and macrophages exposed to silica were treated with different concentrations of BHE.The results of cell viability assay showed that the selected concentrations of BHE had no inhibitory effect on MH-S cells.ELISA assay indicated that BHE could effectively inhibit the secretion of IL-1?,IL-6,TNF-? and MCP-1 by macrophages exposed to silica(P <0.01).Compared with macrophages exposed to silica alone,BHE restrained the phosphorylation level of p38 and JNK in a dose-dependent manner(P <0.001),while BHE did not affect the total p38 and JNK protein levels.Compared with macrophages exposed to silica alone,BHE reduced the expression of NF-?B in macrophage nuclei(P <0.0001).Flow cytometry results showed that silica could induce the apoptosis of MH-S cells,while BHE could decrease the proportion of MH-S cells apoptosis(P <0.0001).Compared with macrophages exposed to silica alone,BHE downregulated the expression of cleaved Caspase-3 and Bax and upregulated the level of PARP and Bcl2(P <0.0001).BHE reduced the expression of i NOS and increased the expression of Nrf2 and HO-1 in macrophages exposed to silica(P <0.01).BHE activated Nrf2/HO-1 signaling,inhibited SP-induced activation of MAPK signaling and alleviated silica-induced inflammation through restraining the oxidative stress response.To investigate the effect of BHE on silica-injured mice,we established an experimental silicosis mouse model,the mice were gavage with different concentrations of BHE.HE,immunohistochemistry and Giemsa staining showed that,compared with the mice exposed to silica alone,BHE reduced the accumulation of inflammatory cells in the lungs.BHE decreased the levels of IL-1?,IL-6,TNF-? and MCP-1 in the BALF of mice exposed to silica(P <0.05).Masson staining,q PCR and hydroxyproline indicated that BHE could downregulate the deposition of collagen ? and the expression of fibronectin in the lungs(P <0.05).Compared with the mice exposed to silica alone,BHE could lessen the proportion of Th1,Th2,Th17 and Tregs cells in the hilar lymph nodes of mice(P <0.05).Western blot analysis showed that BHE decrease the phosphorylation of p38 and JNK protein in a dose-dependent manner(P <0.01),while BHE did not affect the total p38 and JNK protein levels.BHE reduced the expression of i NOS(P <0.01),while added the expression of Nrf2 and HO-1 in the lung(P <0.01).In summary,we found that treatment with polyphenolrich BHE alleviated silica-induced lung inflammation and fibrosis by modulating the oxidative stress response,alleviating macrophages and Th immune responses and altering Nrf2/HO-1-related MAPK signaling.These represent the first demonstrations of such properties associated with BHE treatment and offered insight into the mechanisms associated with their immune-regulatory effects.