Interleukin-10 Does Not Contribute To The Protective Effects Of Anisodamine On Septic Shock In Mice

INTRODUCTIONSepsis is a major cause of morbidity and mortality in hospitals. A severe and uncontrolled systemic inflammatory response triggered by an invading microbe may lead to evolving multi-organ dysfunction,even death.Anisodamine, an alkaloid structurally related to atropine, isolated from the Chinese herb Anisodas tanguticus, is clinically used in China to treat septic, traumatic and hemorrhagic shock. Many studies have shown that anisodamine could significantly reduce the mortality of patients suffering from bacterial meningitis, severe acute pancreatitis and toxic bacillary dysentery[12-14]. Moreover, it also had been reported that anisodamine markedly inhibited pro-inflammatory cytokine production at the transcriptional level. It is therefore logical to contribute this anti-septic shock effect to the inhabitation of inflammation. However, the mechanisms of its anti- inflammation action are not fully understood.Interleukin-10 (IL-10) is a 35-kDa homodimeric protein, produced primarily by monocytes, T cells, and B cells, that inhibits the production of proinflammatory cytokines in vitro. It inhibits the expression of many genes induced by endotoxin, which can prevent the septic shock. So in this study, we aimed to investigate whether the therapeutic effect of anisodamine on the septic shock could—at least in part—be brought about by the activation of IL-10 in mice.METHODSExperiment 1: The effects of anisodamine on the survival time in LPS-induced shock mice Experiment was performed in 120 male KM mice weighed from 18 to 22 g. Mice were injected with LPS 50 mg/kg (i.p.) to produce a lethal endotoxemia. Then all mice were randomized into 6 groups: one control and 5 treatment groups with anisodamine (i.p.): (1) control: rats received an injection of normal saline (i.p.); (2) anisodamine 40mg/kg; (3) anisodamine 80mg/kg; (4) anisodamine 120mg/kg; (5) anisodamine 160mg/kg; (6) anisodamine 200mg/kg (n=20 in each group). The survival time was recorded over 24 hours.Experiment 2 The effects of anisodamine on the survival time in LPS-induced shock mice with IL-10 KO Experiment was performed in 40 mice with IL-10 KO weighed from 25 to 30 g. They were injected with LPS 200 mg/kg (i.p.) to produce a lethal endotoxemia. Then all mice were randomized into 2 groups (n=20 in each group): (1) control group: rats received an injection of normal saline (i.p.); (2) treatment group: rats received an injection of anisodamine 150mg/kg (i.p.). Then the survival time was recorded over 24 hours.Experiment 3 The effects of anisodamine on the expression of IL-10 in macrophages pretreated with LPS RAW264.7 cells were grown in DMEM (GibcoBRL, Grand Island, NY), supplemented with 10% FBS (Hyclone, Logan, UT), 50uM mercaptoethanol, 20mM HEPES, 10mM sodium pyruvate and 50ug/ml gentamicin (GibcoBRL, Grand Island, NY). Then cells were dispensed in six-well plates at 5×10~4 cells per well, and then cultured for 18 h. This culture then went (1)untreated (control) or pre-treated with (2) LPS (1mg/L) alone; (3) anisodamine alone (100mg/L); (4) LPS + anisodamine (1 +10mg/L); (5) LPS + anisodamine (1 +50mg/L); (6) LPS + anisodamine (1 +100mg/L); and (7) LPS + anisodamine (1 +500mg/L). The cells were incubated at 37℃for 4 hours. Then the cell culture supernatants were collected for the measurement of IL-10 using Enzyme-linked immunosorbent assay (ELISA) kit. The experiments were repeated for 3 times and all samples were run in duplicate.REULTSExperiment 1: The effect of anisodamine on the survival time induced by LPS in KM mice There existed an obvious difference between control group and 5 dosages treatment groups with anisodamine (Log-Rank testingχ~2=21.7, P<0.001). In control group, the morality was 80% within 24 hours, and other treatment groups with anisodamine 40, 80, 120, 160, 200 mg/kg was 45%, 40%, 10%, 40%, 55%, respectively. From the dosage of 40 to 120 mg/kg, anisodamine obviously increased the 24 hours survival rate from 45 to 90% with a dosage dependent way, while from the dosage of 120 to 200 mg/kg, the increase of survival rate was decrease from 90% to 45% also with a dosage dependent way.Experiment 2 The effect of anisodamine on the survival time induced by LPS in IL-10 Knock out (KO) mice There existed an obvious difference between control group and treatment group with anisodamine (150 mg/kg) in IL-10 KO mice (Log-Rank testingχ2=4.59, P<0.05). In control group, the morality was 50% within 24 hours while 20% in anisodamine group. The survival rate was significantly increased by anisodamine (from 50% to 80%).Experiment 3 The effect of anisodamine on the expression of IL-10 induced by LPS in macrophages Results are shown in Fig 3. Cultured RAW264.7 cells produced small amounts of IL-10. Incubation these cells with LPS 1 mg/L and anisodamine (100 mg/L) alone had no significant influence on the expression of IL-10. Co-incubation with LPS (1 mg/L) and different concentrations of anisodamine decreased the secretion of IL-10 with a dosage dependent way (from 10 to 50 mg/L). And compared with LPS 1mg/L group, the decrease was significant at the dosage of 100 (P<0.05) and 500 mg/L (P<0.001).CONCLUSIONIn conclusion, within a special dosage range, anisodamine could significant increase the survival time of septic shock mice with a dosage dependent way. This effect might be not dependent on the expression on IL-10.