Downregulation of TLR4/MyD88/p38MAPK and JAK/STAT pathway in RAW 264.7 cells by Alpinia galanga reveals its beneficial effects in inflammation

Alpinia galanga, commonly known as greater galangal or raasna, is widely used in Ayurveda against various inflammatory disorders. It is also known as Kulinjan, Aratha, Rasna or Sugandhamula. Some of the Ayurvedic preparations using the rhizome of Alpinia galanga are Rasnadi kashayam, Rasna panchakam, Rasnapthakam, and Rasnarendadi. The aromatic rhizome is the source of the drug greater galangal and it is also used as a spice in South and South East Asia. However, the molecular mechanism of action of A galanga against inflammation remains poorly understood.To elucidate the anti-inflammatory effect of hydroalcoholic extract of Alpinia galanga rhizome.The mechanism of the anti-inflammatory effect of hydroalcoholic extract of Alpinia galanga (AGE) was investigated by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunofluorescence in LPS stimulated murine macrophage cell line (RAW 264.7). HPLC analysis was done to elucidate the rich polyphenolic nature of AGE.The study showed that pre-treatment with AGE downregulated the release of pro-inflammatory mediators (IL-6, TNF-α, NO, and ROS) and stimulated the release of anti-inflammatory mediator IL-10 in LPS stimulated RAW 264.7 cells. The vital enzymes of inflammation (iNOS, COX-2, and MMP-9) were also downregulated by pre-treatment with AGE. AGE targeted the upstream elements of the inflammatory cascade by blocking LPS induced activation of TLR4 and JAK/STAT pathway. The phosphorylation of downstream kinases was significantly affected. The inhibition of nuclear translocation of NFκB further confirmed the specific inhibition of the TLR4 pathway. Particularly AGE inhibited the phosphorylation of JNK, p38, IκBα, and STAT. HPLC analysis of the AGE showed the polyphenol-rich nature of the extract.The results from this study provide firm evidence that AGE exerts its anti-inflammatory effect via modulation of TLR4 and JAK/STAT pathway.