Effects of LAMC2 on macrophage polarization and regulation of NF-κB and ERK1/2 signaling pathways
10.3760/cma.j.cn431274-20231026-00459
- VernacularTitle:LAMC2对巨噬细胞极化及NF-κB和ERK1/2信号通路调控的作用
- Author:
Zhenzhong JI
1
;
Zhengguo HU
Author Information
1. 武汉大学中南医院老年医学科，武汉 430071
- Keywords:
Diabetic nephropathy;
Macrophages polarization;
LAMC2;
NF-κB signaling pathway;
ERK signaling pathway
- From:
Journal of Chinese Physician
2024;26(11):1669-1676
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the role and potential mechanism of middle layer adhesion γ2 (LAMC2) in the regulation of macrophage polarization in diabetic nephropathy (DKD).Methods:Using GEO database, LAMC2 was identified as the main research object. Mouse mononuclear macrophages RAW264.7 were cultured in vitro and divided into the following 6 groups: Control group (blank control group), HG group (high glucose group), Treated with 30 mmol/L glucose, si-NC group (treated with 30 mmol/L glucose and transfected with negative control), si-LAMC2 group -1# (treated with 30 mmol/L glucose and transfected with shRNA-LAMC2 construct 1), si-LAMC2 group -2# (treated with 30 mmol/L glucose and transfected with SHRnA-LAMC2 construct 1), and SI-LAMC2 group-2# (treated with 30 mmol/L glucose) Group 3# of si-LAMC2 was treated with mmol/L glucose and transfected with shRNA-LAMC2 construct 2, and Group 3# of Si-LAMC2 was treated with 30 mmol/L glucose and transfected with shRNA-LAMC2 construct 3. Real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) and Western Blot assay were used to detect LAMC2 expression in macrophages, small interfering RNA (siRNA) technique was used to inhibit LAMC2 expression, cell proliferation was evaluated by EdU staining, and cell invasion ability was evaluated by Transwell assay. The expressions of inducible nitric oxide synthase (iNOS), interleukin (IL)-12, Tumor necrosis factor-α (TNF-α), Arg-1, CD163 and IL-10 were detected by qRT-PCR, the expressions of CD86 and CD206 were detected by cellular immunofluorescence staining, and the expressions of key proteins in nuclear factor-κB (NF-κB) and extracellular signal regulated kinase (ERK) signaling pathways were detected by western blot assay.Results:Gene analysis in GEO database showed that the expression of LAMC2 in DKD and M1 macrophages was significantly up-regulated (all P<0.01). Compared with the blank Control group, the mRNA and protein expression of LAMC2 in macrophages of HG group were higher (all P<0.01). EdU staining and transwell statistics showed that, the EdU positive rate in HG and si -NC groups was lower than that in the Control group, and the number of invasive cells was significantly higher than that in the Control group (all P<0.05). The EdU positive rate of the si-LAMC2 group was higher than that of the si-NC group, and the number of invaded cells was significantly lower than that of the si-NC group (all P<0.05). Compared with Control group, the mRNA expression levels of M1 macrophage markers iNOS, IL-12 and TNF-α were higher in HG group and si-NC group (all P<0.01). Compared with the si-NC group, the mRNA expression levels of the above three markers in the si-LAMC2 group were lower (all P<0.01). Compared with the Control group, the mRNA expression levels of M2 macrophage markers Arg-1, CD163 and IL-10 in HG and si-NC groups were significantly decreased (all P<0.01). Compared with the si-NC group, the mRNA expression levels of the above three markers in the si-LAMC2 group were higher (all P<0.01). Compared with the Control group, the fluorescence intensity of CD86 in HG and si-NC groups was significantly higher, and the fluorescence intensity of CD206 was lower. Compared with the si-NC group, the fluorescence intensity of CD86 in the si-LAMC2 group was significantly lower, and CD206 were higher. Compared with the Control group, the expression levels of p-ERK1/2 and p-NF-κBP65 in HG and si-NC groups were significantly higher (all P<0.01). Compared with the si-NC group, the expression levels of two proteins in the si-LAMC2 group were significantly lower (all P<0.01). Conclusions:LAMC2 may play important physiological and pathological functions in diabetic nephropathy by promoting the polarization of macrophage M1 and affecting NF-κB and ERK1/2 signaling pathways.
