Effect of zingiber officinale rhizome extracts and [6]-gingerol on the activity and expression of mmp-2,-9 and timp-1, 2 involved in vascular leakage in an in vitro model of dengue virus-3 infection}

Abstract

Severe dengue following dengue virus (DV) infection is caused by increased vascular permeability leading to hypovolemic shock. The role of matrix metalloproteinases (MMPs) and their regulation by tissue inhibitor of metalloproteinases (TIMPs) have been established in vascular permeability. Control of MMPs and TIMPs activity is of great significance to prevent vascular permeability caused by DV infection. Zingiber officinale Roscoe (ZO), is one of the most frequently and heavily consumed dietary condiments with anti-oxidant, anti-inflammatory, immunomodulatory and anti-cancer properties. This research investigated the effects of ZO rhizome extracts and pure [6]-gingerol on the activity and expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 to ameliorate leakage induced by DV infection using an in vitro model of DV infection. Total phenolic contents in terms of mg gallic acid equivalents/ gram for methanol (ZOM) and aqueous extract (ZOA) were 252.89 ± 0.56, 68.17±0.28 and possessed 137.32 ± 2.47, 29.32±1.97 mg [6]-gingerol content per gram of extract were determined using high performance liquid chromatography. The median inhibitory concentration (IC50) value of ZOM, ZOA were 221.5μg/mL, 348.8μg/mL for Vero cells and 186.4, 368.1μg/mL, respectively for MDCK cells as revealed by the MTT assay. The activity of MMP-2 and to a lesser extent MMP-9 were significantly enhanced in the conditioned media (CM) collected from the dengue virus infected Vero cells compared to CM from non-infected cells (p<0.0001). The activities of MMP-2 and MMP-9 were significantly inhibited following treatment with ZOM, [6]-gingerol and ZOA in concentration dependent manner as demonstrated by zymography. mRNA expression of TIMP-1 and TIMP-2 were significantly upregulated accompanied by the downregulation of mRNA expression of MMP-2 and MMP-9 by ZOM, pure [6]-gingerol and ZOA in a dosedependent manner as demonstrated by qReal time RT-PCR in dose dependent manner. The in vitro permeability assay showed that CM from DV-infected Vero cells contain soluble and active factors like MMPs favoring increase of MDCK monolayer permeability compared to CM collected from non-infected cells (p<0.0001). ZOM, [6]-gingerol and ZOA reduced the permeability caused by MMPs in a dose dependent manner. The results of this study suggest that Zingiber officinale rhizome extracts and pure [6]-gingerol may be effective in the control of DV-induced permeability through the reduction of activities and expression of proteases which degrade the adhesion molecules between cells. This may provide the basis for developing new and effective methods in controlling severe dengue complications that warrant further investigation.