Experimental Study On Protective Effects Of Total Flavonoids Extracted From Trollius Altaicus Bunge On Acute Bronchial Epithelial BEAS-2B Cell Injury Induced By PM_2.5

Objective:To investigate the protective effect of total flavonoids of Trollius altaicus Bunge onPM_2.5-induced acute bronchial epithelial BEAS-2B cells and its possible mechanism.Methods:?1?The CCK-8 method was used to draw the growth curve of BEAS-2B cells and to find the logarithmic growth phase of the cells.?2?The CCK-8method was used to determine the hemi-inhibitory concentration(IC₅₀)of total flavonoids of Trollius altaicus Bunge on BEAS-2B cells.?3?The CCK-8 method was used to detect the effect of PM_2.5.5 administration with different concentrations on the viability of BEAS-2B cells.?4?They were divided into the blank control group,PM_2.5.5 model group,PM_2.5+total flavonoids of Trollius altaicus Bunge high,medium and low dose(28.19,14.10,7.05mg·L^-1)groups,and PM_2.5+dexamethasone(1mmol·L^-1)positive control group,a total of 6 groups.At the beginning of the experiment,each dose group of the test substance and the positive control group respectively intervened in cell culture according to the set drug dose for 24 hours before constructing model;After 24 hours,except the blank control group,in the remaining groups,PM_2.5.5 solution at 100 mg·L^-11 all acted on BEAS-2B cells for 24 hours.After the experiment,the morphological changes of each group were observed under microscope,the cell and culture supernatants were collected respectively,and the cell culture supernatants of each group were used.The ELISA method was used to determine the contents of IL-6,IL-8,IL-1?,and TNF-?.The Colorimetry method was used to detect the content of superoxide dismutase?SOD??malondialdehyde?MDA?,lactate dehydrogenase?LDH?and glutathione?GSH?;qRT-PCR method was used to detect mRNA contents of IL-6,IL-8,IL-1?,TNF-?;Western Bolt method was used to detect the expression levels of proteins p65,IKK-?,IKB-?.Results:?1?The logarithmic growth phase of BEAS-2B cells was 3 to 5 days.?2?The the hemi-inhibitory concentration IC₅₀0 of total flavonoids of Trollius altaicus Bunge on BEAS-2B cells was 281.9mg·L^-1.?3?The administrated dose of PM_2.5.5 was 100mg·L^-1.?4?The cell morphology of each group was observed under the microscope.The cell morphology of the normal control group was oval and the cell arrangement was tight.Compared with the normal control group,the cells in the PM_2.5.5 model group were shrunken and deformed.The intercellular space was significantly increased,and the connections were sparse.Compared with the PM_2.5.5 model group,the deformation degree of cell morphology of the PM_2.5+total flavonoids of Trollius altaicus Bunge high and medium dose groups was significantly reduced.Compared with the PM_2.5+dexamethasone positive control group,the deformation degree of cell morphology of PM_2.5+total flavonoids of Trollius altaicus Bunge high and medium dose groups was significantly reduced.However,Compared with the PM_2.5+dexamethasone positive control group,the deformation degree of cell morphology of the PM_2.5+total flavonoids of Trollius altaicus Bunge low dose group was not significantly different.Among them,between PM_2.5+total flavonoids of Trollius altaicus Bunge high,medium and low dose groups,with the increase of drug concentration,the deformation degree of cell morphology also decreased.?5?Compared with the normal control group,the SOD and GSH contents of the PM_2.5model group were significantly reduced?P<0.05?,and the MDA and LDH contents were increased?P<0.05?;Compared with the PM_2.5.5 model group,the contents of SOD and GSH of PM_2.5+total flavonoids of Trollius altaicus Bunge high,medium and low dose groups increased,and the MDA and LDH were all decreased?P<0.05?;Compared with the PM_2.5+dexamethasone positive control group,the contents of SOD and GSH of the PM_2.5+total flavonoids of Trollius altaicus Bunge high and medium dose groups increased,and the MDA,LDH contents were all reduced?P<0.05?;Between PM_2.5+total flavonoids of Trollius altaicus Bunge high,medium and low dose groups,with increase of the drug concentration,the SOD and GSH content also increased,?P<0.05?,the MDA and LDH content were also reduced?P<0.05?.?6?Compared with the normal control group,the contents of IL-1?,IL-6,IL-8,TNF-?in the cell culture supernatants and intracellular mRNA contents of IL-1?,IL-6,IL-8 and TNF-?of the PM_2.5.5 model group increased significantly?P<0.05?;Compared with PM_2.5.5 model group,the contents of IL-1?,IL-6,IL-8,TNF-?in the cell culture supernatants and intracellular mRNA contents of IL-1?,IL-6,IL-8 and TNF-?of the PM_2.5+total flavonoids of Trollius altaicus Bunge high,medium and low dose groups were all reduced?P<0.05?;Compared with PM_2.5+dexamethasone positive control group,the contents of IL-1?,IL-6,IL-8,TNF-?in the cell culture supernatants and intracellular mRNA contents of IL-1?,IL-6,IL-8 and TNF-?of the PM_2.5+total flavonoids of Trollius altaicus Bunge high and medium dose groups were all significantly reduced?P<0.05?;Between PM_2.5+total flavonoids of Trollius altaicus Bunge high,medium and low dose groups,with increase of the drug concentration,the contents of IL-1?,IL-6,IL-8,and TNF-?in the cell culture supernatants also decreased?P<0.05?,and intracellular mRNA contents IL-1?,IL-6,IL-8,and TNF-?also decreased?P<0.05?.Western Bolt test results showed that compared with the normal control group,the expression levels of p65 and IKK-?in the PM_2.5.5 model group significantly increased,and the expression level of IKB-?significantly decreased?P<0.05?;Compared with the PM_2.5.5 model group,the expression levels of p65 and IKK-?of the PM_2.5+total flavonoids of Trollius altaicus Bunge high,medium and low dose groups slightly decreased,and the expression level of IKB-?slightly increased?P<0.05?;Compared with the PM_2.5+dexamethasone positive control group,the expression levels of p65,IKK-?of the total flavonoids of Trollius altaicus Bunge high and medium dose groups slightly decreased,the expression level of IKB-?slightly increased?P<0.05?;The expression levels of p65 and IKK-?between the the PM_2.5+total flavonoids of Trollius altaicus Bunge high,medium and low dose groups also decreased with increase of the drug concentration and the expression level of IKB-?increased?P<0.05?.Conclusion:Total flavonoids of Trollius altaicus Bunge can regulate the expression levels of downstream inflammatory cytokines and reduce inflammation damage by inhibiting apoptosis,reducing oxidative stress injury,inhibiting the secretion of inflammatory cytokines and inhibiting the PM_2.5-induced activation of NF-?B signal pathway.Thus,it plays a protective role in PM_2.5-induced BEAS-2B cells acute injury.