In Vitro Study On Anti-endotoxin Effect Of Xuebijing Injection

Endotoxin,also called lipopolysaccharides(LPS),is a major component in outer membrane of the Gram-negative bacteria.Endotoxemia(ETM) participates in the onset and development of lots of diseases,such as sepsis, burn,acute respiratory distress syndrome(ARDS),acute and chronic liver diseases,necrotic pancreatitis and so on,even leads to septic shock, disseminated intravascular coagulation(DIC) and multiple organ dysfunction syndrome(MODS).Now,Monocytes and macrophages are considered to be the targets of endotoxin,which are the effector cells of endotoxin tolerance as well.And Toll-like receptor 4(TLR4) plays a vital role in endotoxin signaling;Meanwhile the Interleukin-1 receptor-associated kinase-M(IRAK-M) is a important negative-regulator in the signaling.Xuebijing injection is purified from the traditional Chinese herbs of Safflower,Radix paeoniae rubra,Ligustici chuanxiong,Salvia miltiorrhiza and Angelica.It is reported that Xuebijing has the effects on direct anti-andotoxin and reducing the release of cytokine out of control,and also can ameliorate microcirculation,protect and repair the organ impaired in stress;it may decrease the mortality of sepsis and MODS.Based on these,the study is carried out to objectively evaluate the anti-endotoxin and anti-inflammatory effects of Xuebijing.PartⅠIn vitro study on the neutralizing or anti-endotoxin effect of XuebijingObjective:To understand whether Xuebijing has the neutralizing and anti-endotoxin effect or not.Methods:The standard endotoxin was mixed with different concentrations of Xuebijing and heated in the water bath,then the concentration of endotoxin in these mixtures were detected by Limulus test (Chromogenic substrate method).Results:Compared to the positive control,level of the endotoxin was higher when the concentration of Xuebijing was added;But each concentration group of Xuebijing,together with the positive control,had lower endotoxin level in the mixtures when time went on;However,there was no significant difference among all the groups(p＞0.05).Conclusion:Xuebijng has no effect on neutralizing or anti-endotoxin in vitro.PartⅡEffcets of Xuebing on the THP-1 cell line challenged by endotoxinObjective:To evaluate the effect of Xuebijing on blocking the release of TNF-αfrom the THP-1 cell line challenged by endotoxin.And trying to probe the probable mechanism whether is related to the blockage of endotoxin signaling or endotoxin tolerance.Methods:The THP-1 cell line were challenged by 10ng/ml endotoxin for 4 hours,which were protected by Xuebijing in different concentrations(10,25 and 50mg/ml respectively) and time(4,12 and 24 hours respectively) before. The concentration of TNF-αin culture supernatants was detected by ELISA assay.Total RNA were extracted from each group,then the Real-Time PCR technique was used to estimate the expression of TLR4 and IRAK-M mRNA.Results:(1)There was no significant difference among the concentration of TNF-αfrom all the groups(primed at different concentrations of Xuebijing and for different time)(p＞0.05).(2)Only primed for 24 hours,TLR4 mRNA was a little up-regulated when the concentration of Xuebijing was added;however, compared to the control,there was no significant difference(p＞0.05).But at the concentration of 50mg/ml,TLR4 mRNA was 1.547-fold increase in 24 hours group compared to 4 hours group(p＜0.05).(3) Primed for 4 and 12 hours, there was no significant difference of IRAK-M mRNA between each concentration group of Xuebijing and control;but when primed for 24 hours, IRAK-M mRNA was 1.349-fold increase in 50mg/ml Xuebijing group compared to control(p＜0.05);but there was no significant difference among other groups; And in 50mg/ml Xuebijing group,IRAK-M was up-regulated when time went on, however,there was no significant difference among them(p＞0.05).Conclusion:(1)Xuebijng has no effect on blocking the release of TNF-αfrom the THP-1 cell line challenged by endotoxin.(2)When primed at high concentration of Xuebijing and for long time,the expression of both TLR4 and IRAK-M mRNA may be up-regulated,but the meaning is not clear yet.