Role Of α₂ Adrenoceptor In The Protective Effect Of Berberine Against Endotoxemia In Mice

ObjectiveTo observe the effect of yohimbine, aα² adrenoceptor antagonist, on protection of berberine agaist endotoxemia, and further investigate the possible mechanisms.MethodsFirstly, we examined the effects of berberine in combination with yohimbine on the mortality of mice suffered from LPS (20 mg/kg) or CLP. Secondly, lung, kidney, liver and small intestine were collected for the HE staining at 12 h after LPS administration in mice, and then we observed the effect of berberine in combination with yohimbine on histological changes of the above organs in mice challenged with LPS; the serum was collected in order to assay the liver and kidney function at the same time. Thirdly, we investigated the effect of berberine in combination with yohimbine on serum tumor necrosis factor-α(TNF-α), interleukin-12 (IL-12), IL-1β, IL-10, IL-6 and interferon-γ, (IFN-γ) contents with enzyme-linked immunosorbent assay (ELISA) in mice challenged LPS, and measured nitric oxide (NO) content in serum with the technique of nitrate reductase. Lastly, we observed the protective effect of berberine in combination with yohimbine against LPS-induced lung injury. The mice were sacrificed at 12h after LPS injection, and the right lung was used to determine the ratio of wet to dry lung tissue weight (W/D). The total protein, the amounts of white blood cells (WBC), polymorphonuclear neutrophils (PMN), thromboxane B₂ and leukotriene in bronchoalveolar lavage fluid (BALF) were determined. The expression and phosphorylation of cytosolic phospholipase A₂ (cPLA₂) in the lung tissue were also detected with Western Blotting.Results1. Pretreatment with yohimbine remarkably enhanced the effect of berberine against endotoxemia, the combination of berberine and yohimbine reduced the histological injury of lung, liver, kidney and small intestine, and significantly increased the survival rate of mice challenged with LPS or CLP. Pretreatment with 2mg/kg yohimbine in combination with 50mg/kg berberine for 3 days decreased the mortality of endotoxemic mice by 80% at 6 d after LPS challenge.2. There was a distinct decrease in serum TNF-α, IL-1βand NO contents and increase in serum IL-10, IL-12p40 and IFN-γ, production in berberine treatment group compared with LPS group, yohimbine did not block the effect ofberberine on TNF-α, NO and IFN-γ,, but reversed the effect of berberine on IL-1βand IL-12 p40, and promoted berberine-induced IL-10 production in endotoxemic mice. In addition, yohimbine in combination with berberine increased serum IL-6 level in endotoxemic mice.3. The pretreatment with berberine significantly attenuated lung injury induced by LPS, the expression and phosphorylation of cytosolic phospholipase A₂ (cPLA₂) in the lung tissue, the content of thromboxane B₂ in the BALF were lower than those in LPS group. Berberine combination with yohimbine also ameliorated LPS-induced lung injury, there was no marked difference in above parameter compared with berberine treatment group.ConclusionYohimbine remarkably enhances the effect of berberine against endotoxemia. Berberine decreasing TNF-α, NO production and increasing IL-10, IFN-γ, release are independent ofα₂ adrenoceptor, while inhibiting IL-1βrelease and promoting IL-12 production dependent onα₂ adrenoceptor in endotoxernic mice. Moreover, Berberine attenuates LPS-induced lung injury via inhibiting cPLA₂ expression and activation in aα₂ adrenoceptor-independent manner. These data indicate berberine-inducedα₂ adrenoceptor activation inhibits its protection against endotoxemia, berberine combined with yohimbine might provide an effective therapeutic approach to endotoxemia.