Infiltration of activated neutrophils into the vital organs contributes to the multiple organ dysfunctions in sepsis. In the present study, we investigated the effects of berberine in combination with yohimbine (BY) on neutrophil tissue infiltration and multiple organ damage during sepsis, and further elucidated the involved mechanisms. Sepsis was induced in mice by cecal ligation and puncture (CLP). BY or CCR2 antagonist was administered 2h after CLP, and anti-IL-10 antibody (IL-10 Ab) or control IgG was injected intraperitoneally just before BY treatment. We found that IL-10 production was enhanced by BY therapy in septic mice. BY significantly attenuated neutrophil tissue infiltration and multiple organ injury in CLP-challenged mice, all of which were completely reversed by IL-10 Ab pretreatment. The levels of KC, MCP-1, MIP-1α and MIP-2 in the lung, liver and kidney were markedly increased 6h after CLP. BY reduced the tissue concentrations of these chemokines in septic mice, but IL-10 Ab pretreatment did not completely eliminate these inhibitory effects of BY. Particularly, dramatically increased CCR2 expression in circulating neutrophils of septic mice was reduced by BY and this effect was completely abolished by IL-10 Ab pretreatment. Furthermore, CCR2 antagonist also inhibited lung and renal injury and neutrophil infiltration in septic mice. Taken together, our data strongly suggest that BY therapy attenuates neutrophil tissue infiltration and multiple organ injury in septic mice, at least in part, via IL-10-mediated inhibition of CCR2 expression in circulating neutrophils.

Myocardial depression is an important contributor to mortality in sepsis. We have recently demonstrated that α2-adrenoceptor (AR) antagonist, yohimbine (YHB), attenuates lipopolysaccharide (LPS)-induced myocardial depression. However, the mechanisms for this action of YHB are unclear. Here, we demonstrated that YHB decreased nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) levels in the myocardium and plasma, attenuated cardiac and hepatic dysfunction, but not kidney and lung injuries in endotoxemic mice. Immunohistochemical analysis revealed that cardiac α2A-AR was mostly located in sympathetic nerve presynaptic membrane; YHB decreased cardiac α2A-AR level and promoted cardiac norepinephrine (NE) release in endotoxemic mice. Reserpine that exhausted cardiac NE without markedly decreasing plasma NE level abrogated the inhibitory effects of YHB on cardiac TNF-α and iNOS expression as well as cardiac dysfunction, but not the suppressive effects of YHB on plasma TNF-α and NO elevation in LPS-challenged mice. Furthermore, both reserpine and YHB significantly inhibited LPS-induced myocardial apoptosis. α1-AR, β2-AR, but not β1-AR antagonists reversed the inhibitory effect of YHB on LPS-stimulated myocardial apoptosis. However, β1-AR antagonist attenuated LPS-caused cardiomyocyte apoptosis, partly abolished the protective effect of YHB on the left ventricular ejection fraction in endotoxemic mice. Altogether, these findings indicate that YHB attenuates LPS-induced cardiac dysfunction, at least in part, through blocking presynaptic α2A-AR and thus increasing cardiac NE release. YHB-elevated cardiac NE improves cardiac function via suppressing cardiac iNOS and TNF-α expression, activating β1-AR and inhibiting cardiomyocyte apoptosis through α1- and β2-AR in endotoxemic mice. However, cardiac β1-AR activation promotes LPS-induced cardiomyocyte apoptosis.

Cardiomyocyte tumour necrosis factor α (TNF-α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)-induced cardiomyocyte TNF-α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS-induced TNF-α production in a dose-dependent manner. α₁- adrenoceptor (AR) antagonist (prazosin), but neither β₁- nor β₂-AR antagonist, abrogated the inhibitory effect of NE on LPS-stimulated TNF-α production. Furthermore, phenylephrine (PE), an α₁-AR agonist, also suppressed LPS-induced TNF-α production. NE inhibited p38 phosphorylation and NF-κB activation, but enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and c-Fos expression in LPS-treated cardiomyocytes, all of which were reversed by prazosin pre-treatment. To determine whether ERK1/2 regulates c-Fos expression, p38 phosphorylation, NF-κB activation and TNF-α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c-Fos expression, p38 mitogen-activated protein kinase (MAPK) phosphorylation and TNF-α production, but not NF-κB activation in LPS-challenged cardiomyocytes. In addition, pre-treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS-induced TNF-α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c-Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF-α production and prevented LPS-provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁-AR by NE suppresses LPS-induced cardiomyocyte TNF-α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF-κB activation.

Cardiomyocyte apoptosis is an important event in doxorubicin (DOX)-induced cardiac injury. The aim of the present study was to investigate the protection of berberine (Ber) against DOX- triggered cardiomyocyte apoptosis in neonatal rat cardiomyocytes and rats. In neonatal rat cardiomyocytes, Ber attenuated DOX-induced cellular injury and apoptosis in a dose-dependent manner. However, Ber has no significant effect on viability of MCF-7 breast cancer cells treated with DOX. Ber reduced caspase-3 and caspase-9, but not caspase-8 activity in DOX-treated cardiomyocytes. Furthermore, Ber decreased adenosine monophosphate-activated protein kinase α (AMPKα) and p53 phosphorylation at 2 h, cytosolic cytochrome c and mitochondrial Bax levels and increased Bcl-2 level at 6 h in DOX-stimulated cardiomyocytes. Pretreatment with compound C, an AMPK inhibitor, also suppressed p53 phosphorylation and apoptosis in DOX-treated cardiomyocytes. DOX stimulation for 30 min led to a loss of mitochondrial membrane potential and a rise in the AMP/ATP ratio. Ber markedly reduced DOX-induced mitochondrial membrane potential loss and an increase in the AMP/ATP ratio at 1 h and 2 h post DOX exposure. In in vivo experiments, Ber significantly improved survival, increased stroke volume and attenuated myocardial injury in DOX-challenged rats. TUNEL and Western blot assays showed that Ber not only decreased myocardial apoptosis, caspase-3 activation, AMPKα and p53 phosphorylation, but also increased Bcl-2 expression in myocardium of rats exposed to DOX for 84 h. These findings indicate that Ber attenuates DOX-induced cardiomyocyte apoptosis via protecting mitochondria, inhibiting an increase in the AMP/ATP ratio and AMPKα phosphorylation as well as elevating Bcl-2 expression, which offer a novel mechanism responsible for protection of Ber against DOX-induced cardiomyopathy.

AIM: To investigate the effects of berberine(Ber) and yohimbine(Y) on gene expression in Toll-like receptor 4(TLR4) signaling pathways in the spleen of endotoxemic mice.METHODS: The male BALB/c mice were randomly divided into the following groups: control,lipopolysaccharides(LPS),Ber+LPS,Y+Ber+LPS,Y+LPS,Ber,Y+Ber and Y alone.The mice were treated with water,Y(2 mg/kg) or/and Ber(50 mg/kg) intragastrically once a day for 3 days,and then injected intraperitoneally with normal saline or LPS(20 mg/kg) 1 h after intragastrical treatment on day 3.One hour after LPS challenge,the expression changes of 84 genes in TLR4 signaling pathways in the spleen of the mice were examined using RT2 ProfilerTM PCR Array.Moreover,the protein expression of suppressor of cytokine signaling(SOCS)1,SOCS3 and IL-1 receptor-associated kinase(IRAK)-M in the spleen was examined using Western blotting.RESULTS: LPS induced the expression of multiple downstream inflammatory mediators in myeloid differentiation factor 88(MyD88)-dependent and independent signaling pathways,such as TNF-α,IL-1α,IL-1β,IL-6,IFN-γ,IFN-β and CXCL10.Ber not only down-regulated LPS-induced mRNA expression of TNF-α,IL-1β,L-1β and IL-6,which were in the downstream of MyD88-dependent signaling pathway,but also reduced LPS-stimulated mRNA expression of IFN-β and CXCL10,which were in the downstream of MyD88-independent signaling pathway(P<0.05).Y markedly inhibited the mRNA expression of IL-1α,IL-1β and IFN-β in the spleen of endotoxemic mice(P<0.05),but did not affect the mRNA expression of TNF-α,IL-6 and CXCL10(P>0.05).Combination of Ber with Y significantly reduced the mRNA expression of IFN-β and CXCL10 in the spleen of the mice challenged with LPS.One hour after LPS challenge,Ber or/and Y did not increase the protein expression of SOCS1,SOCS3 and IRAK-M in the spleen of the mice.CONCLUSION: Pretreatment with Ber and Y down-regulates the expression of some genes in the downstream of MyD88-dependent and MyD88-independent signal pathways in the spleen of endotoxemic mice.The mechanism of these actions is not related with SOCS1,SOCS3 and IRAK-M at protein level.

<正>目的:观察小檗碱对阿霉素性心肌细胞凋亡的影响及作用机制。方法:利用乳鼠心肌细胞和SD大鼠复制阿霉素性心肌损伤模型,测定阿霉素和小檗碱处理后心肌细胞凋亡与相关信号通路的变化。结果:小檗碱减轻阿霉素引起的乳鼠心肌细胞

AIM:To investigate the mechanisms by which paeoniflorin(Pae) attenuates lipopolysaccharide(LPS)-induced acute lung injury in mice.METHODS: Male BALB/c mice were randomly divided into 4 groups: control,LPS,Pae+LPS,and Pae.Mice were administered intragastrically with double distilled water or Pae(20 mg/kg) once a day for 3 days.One hour after intragastrical treatment on the third day,LPS(20 mg/kg) or normal saline was injected intraperitoneally.Twelve hours after LPS challenge,the histological changes of the lung were observed,and histology score was also assessed.The myeloperoxidase(MPO),cytosolic phospholipase A2(cPLA2) and phosphorylated cytosolic phospholipase A2(phospho-cPLA2) in lung tissues were detected by Western blotting.RESULTS: LPS challenge resulted in acute lung injury,activated cPLA2 and increased MPO content in lung.Pretreatment with paeoniflorin significantly attenuated lung injury induced by intraperitoneal injection of LPS.The levels of MPO and phospho-cPLA2 in the lung tissues of the mice in Pae+LPS group were lower than those in LPS group(P0.05).CONCLUSION: Pretreatment with paeoniflorin remarkably reduces LPS-induced acute lung injury through inhibiting phosphorylation of cPLA2 and decreasing neutrophil infiltration in the lung.These findings provide a new strategy for the prevention and treatment of LPS-induced acute lung injury.