Verapamil and tangeretin enhances the M1 macrophages to M2 type in lipopolysaccharide-treated mice and inhibits the P-glycoprotein expression by downregulating STAT1/STAT3 and upregulating SOCS3.

LPS induced sepsis is a complex process involving various immune cells and signaling molecules. Dysregulation of macrophage polarization and ROS production contributed to the pathogenesis of sepsis. PGP is a transmembrane transporter responsible for the efflux of a number of drugs and also expressed in murine macrophages. Natural products have been shown to decrease inflammation and expression of efflux transporters. However, no treatment is currently available to treat LPS induced sepsis. Verapamil and Tangeretin also reported to attenuate lipopolysaccharide-induced inflammation. However, the effects of verapamil or tangeretin on lipopolysaccharide (LPS)-induced sepsis and its detailed anti-inflammatory mechanism have not been reported. Here, we have determined that verapamil and tangeretin protects against LPS-induced sepsis by suppressing M1 macrophages populations and also through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression in macrophages. An hour before LPS (10 mg/kg) was administered; mice were given intraperitoneal injections of either verapamil (5 mg/kg) or tangeretin (5 mg/kg). The peritoneal macrophages from different experimental groups of mice were isolated. Hepatic, pulmonary and splenic morphometric analyses revealed that verapamil and tangeretin decreased the infiltration of neutrophils into the tissues. Verapamil and tangeritin also enhanced the activity of SOD, CAT, GRX and GSH level in all the tissues tested. verapamil or tangeretin pre-treated mice shifted M1 macrophages to M2 type possibly through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression. Hence, both these drugs have shown protective effects in sepsis via suppressing iNOS, COX-2, oxidative stress and NF-κB signaling in macrophages. Therefore, in our study we can summarize that mice were treated with either Vera or Tan before LPS administration cause an elevated IL-10 by the macrophages which enhances the SOCS3 expression, and thereby able to limits STAT1/STAT3 inter-conversion in the macrophages. As a result, NF-κB activity is also getting down regulated and ultimately mitigating the adverse effect of inflammation caused by LPS in resident macrophages. Whether verapamil or tangeretin offers such protection possibly through the inhibition of P-glycoprotein expression in macrophages needs clarification with the bio availability of these drugs under PGP inhibited conditions is a limitation of this study.

Vitexin along with verapamil downregulates efflux pump P-glycoprotein in macrophages and potentiate M1 to M2 switching via TLR4-NF-κB-TNFR2 pathway in lipopolysaccharide treated mice.

The lipopolysaccharide, a microbial toxin, is one of the major causative agents of sepsis. P-gp expression and its functions are altered during inflammation. LPS has been known to impair the functions of P-gp, an efflux transporter. But the effect of LPS on P-gp expression in murine peritoneal macrophages is poorly understood. Molecular docking studies reveal that vitexin is a potent substrate and verapamil a potent inhibitor of P-gp. In the present experimental study, the curative potential of vitexin as a fruit component and verapamil treated as a control inhibitor of P-gp was examined in a murine LPS sepsis model. The effects of vitexin and verapamil on P-gp expression in macrophages correlating with changes in macrophage polarization and associated functional responses during LPS induced sepsis were studied. Peritoneal macrophages of LPS (10 mg/kg body weight) challenged mice exhibited elevated levels of H2O2, superoxide, and NO in parallel with lower antioxidant activity. LPS treatment increased P-gp expression through increased TLR4/expression. However, LPS challenged mice treated with vitexin (5 mg/kg body weight) + verapamil (5 mg/kg body weight) showed higher anti-oxidant enzyme activity (SOD, CAT and GRx) resulting in reduced oxidative stress. This combination treatment also elevated TNFR2, concomitant with down-regulation of TLR4, NF-κB and P-gp expression in murine peritoneal macrophages, resulting in a switch from M1 to M2 polarisation of macrophages and reduced inflammatory responses. In conclusion, combined vitexin and verapamil treatment could be used as a promising therapy to regulate P-gp expression and protection against LPS mediated sepsis and inflammatory damages.