GM1 Induced the inflammatory response related to the Raf-1/MEK1/2/ERK1/2 pathway in co-culture of pig mesenchymal stem cells with RAW264.7

Abstract

Pig-human xenotransplantation can trigger cell-mediated immune responses. We explored the role of gangliosides in inflammation related to immune rejection in xenotransplantation. Co-culture of xenogeneic cells (pig-MSCs and RAW264.7) was used to emulate xenotransplantation conditions. MTT assay results indicated that cell viability was significantly decreased in pADMSCs co-cultured with RAW264.7 cells. GM1 and GM3 were highly expressed in pADMSCs co-cultured with RAW264.7 cells. pADMSCs co-cultured with RAW264.7 cells strongly expressed pro-inflammatory proteins such as COX-2, iNOS, p50, p65, pIκBα, and TNF-α. GM1-knockdown pADMSCs co-cultured with RAW 264.7 cells did not show significantly altered cell viability, but pro-inflammatory proteins were markedly inhibited. Co-culture of pADMSCs with RAW264.7 cells induced significant phosphorylation (p) of JNK1/2 and pERK1/2. However, pERK1/2 and pJNK1/2 were decreased and MEK1/2 and Raf1 were suppressed in GM1-knockdown pADMSCs co-cultured with RAW264.7 cells. Thus, the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways were significantly upregulated in response to increases of GM1 in co-cultured xenogeneic cells. However, the inflammatory response was suppressed in co-culture of GM1-knockdown pADMSCs with RAW264.7 cells via down-regulation of the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways. Therefore, the ganglioside GM1 appears to play a major role in the inflammatory response in xenotransplantation via the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways.

β‐Cypermethrin Alleviated the Inhibitory Effect of Medium from RAW 264.7 Cells on 3T3‐L1 Cell Maturation into Adipocytes

“…In the present study, exposure to RM had cytotoxic effects on 3T3‐L1 cells, indicating that RAW 264.7 cells inhibited cell proliferation and induced cell death in 3T3‐L1 cells. Consistent with our results, cotreatment with conditioned medium from RAW 264.7 cells reduced the cell viability of 4T1 cells, a mouse breast cancer cell line, preventing the proliferation of cancer cells (Hsieh and Wang, ); the cell viability of pig mesenchymal stem cells was also found to be decreased when they were coincubated with RAW 264.7 cells (Kwak et al, ). However, the cytotoxic effects of RAW 264.7‐conditioned medium on 3T3‐L1 cells was not only significantly alleviated by β‐CYP but also largely recovered with NAC cotreatment.…”

Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells

“…4.14. High-Performance Thin-Layer Chromatography (HPTLC) HPTLC (High-Performance Thin-Layer Chromatography) analysis was conducted following established procedures as detailed in prior study [55]. Purified gangliosides dissolved in chloroform/methanol (1:1, v/v) were meticulously spotted using a capillary tube onto HPTLC plates measuring 10 × 10 cm (Merck Millipore, Darmstadt, Germany).…”

7,8-Dihydroxyflavone induces mitochondrial apoptosis and down-regulates the expression of ganglioside GD3 in malignant melanoma cells

“…Ganglioside was prepared as previously described [47]. Brie y, total lipids were extracted using chloroform/methanol ( which are commonly used as the control cell line, was 214.1 µM (Fig.…”

“…The eluted gangliosides were dried under N 2 gas at 30°C. Finally, the dried samples were stored at -80 ℃ until further experiments.2.13 High-performance thin-layer chromatography (HPTLC)HPTLC analysis was performed as previously described[47]. Puri ed gangliosides in chloroform/methanol (1:1, v/v) were spotted using a capillary tube on 10 × 10 cm TLC plates (Merck Millipore, MA, USA) and then developed with 100 mL chloroform/methanol/0.25% CaCl2 •H 2 O (50:40:10, v/v/v).…”