Amphiregulin Promotes Airway Smooth Muscle Contractile Protein Expression Through The EGFR-ERK1/2 Pathway

Background:Amphiregulin(AREG)expressed in a variety of tissues(lung tissue,airway)and inflammatory cells(leukocyte population,lymphocytes,regulatory T cells and a part of tissue resident regulatory CD4~+T cells)can participate in the repair and reconstruction of damaged tissues,and play a role in regulating inflammation and maintaining homeostasis.Has become a biomarker of asthma.Objective:This study aims to explore the role and mechanism of AREG as an important signal molecule in stimulating the contraction of Airway Smooth Muscle cells(ASMC)by stimulating EGFR on the surface of Airway Smooth Muscle cells(ASMC),so as to provide a theoretical basis for illustrating the role of AREG in Airway contraction.In order to provide a new target for the prevention and treatment of asthma airway hyperresponsiveness.Methods:The primary airway smooth muscle cells of mice were isolated and cultured,purified and identified by immunofluorescence staining.Smooth muscle cells were divided into control and experimental groups(experimental groups were divided into:50ng/ml group,100ng/ml group,200ng/ml group and 400ng/ml group according to the concentration of recombinant AREG(Recombinant amphiregulin,r RNA)treatment),and after 12 hours,the expression of EGFR,ERK1/2and MLC20 at the m RNA level was measured by RT-q PCR;the changes in the expression of EGFR,ERK1/2 and MLC20 and phosphorylated EGFR,ERK1/2 and MLC20 were detected by Western blot.Results:1.Airway smooth muscle cells of mice were isolated successfully.2.At m RNA level,EGFR expression in 200ng/ml and400ng/ml groups was(1.53±0.16,P<0.01),(1.44±0.14,P<0.05)significantly increased compared with the control group;The expression of ERK2 in 200ng/ml and 400ng/ml groups was(1.32±0.11,P<0.05),(1.35±0.05,P<0.01)significantly increased compared with the control group;The expression of MLC20 in 400ng/ml group was(1.18±0.10,P<0.05)significantly increased compared with the control group.3.Western Blot analysis of mouse airway smooth muscle cells(m ASMC)showed that after 12 hours of r AREG intervention,the protein expression levels of m ASMC p-EGFR/EGFR in100ng/m L,200ng/m L and 400ng/m L groups were 1.12±0.01,1.26±0.04 and1.28±0.02 times of those in the control group,respectively,the differences were statistically significant(P<0.05);The protein expression levels of m ASMC p-ERK1/2/ERK1/2 in 200ng/m L and 400ng/m L groups were 1.30±0.14 and 1.31±0.12times that of the control group,respectively,the difference was statistically significant(P<0.05);The protein expression levels of m ASMC p-MLC20/MLC20 in 100ng/m L,200ng/m L and 400ng/m L groups were 1.24±0.09,1.33±0.15 and 1.35±0.13times that of the control group,respectively,the difference was statistically significant(P<0.05).Conclusion:AREG may promote airway smooth muscle contraction by upregulating the phosphorylation level of the receptor EGFR on airway smooth muscle cells,signaling into the cell,leading to increased expression of ERK1/2 protein phosphorylation and increased expression of MLC20 phosphorylation levels of proteins associated with cell contraction,possibly thereby promoting airway smooth muscle contraction.AREG promotes a concentration-dependent elevation of MLC20phosphorylation in airway smooth muscle cells.