## 3 Results
### MSF Suppressed LPS-Induced Septic Shock in Mice  
The anti-inflammatory effects of MSF were examined using endotoxin shock induced in mice by LPS. All mice intraperitoneally (i.p.) injected with 10-40 mg/kg LPS died within 48 h, but 25% of the mice survived after 10 mg/kg LPS injection (Figure 6A). No mortality was observed for mice injected with various concentrations of MSF (Figure 6B). Interestingly, pre-injection of MSF (400 ug/mouse or 4 mg/mouse) in mice 24 h before injection of 20 mg/kg LPS improved the survival rate compared with that of mice that only  
Figure 5: MSF-mediated TNF-\(\alpha\) inhibition was associated with negative regulators in the LPS-TLR4 pathway. THP-1 cells were pretreated with MSF at a concentration of 200 ug/mL for 18 h and/or stimulated by 500 ng/mL LPS for 4 h. mRNA levels of suppressor of cytokine signaling 1 (SOCS-1) (**A**) and tumor necrosis factor \(\alpha\)-induced protein 3 (TNFAIP3/A20) (**B**) in total proteins of the cells were then measured by real-time PCR. (**C**) The levels of SOCS-1 and A20 were quantified by western blot. (**D**) THP-1 cells were pretreated with 25 nM of siRNA of the control, SOCS-1 and/or A20, respectively, for 36 h. The cells were then treated with 200 ug/mL MSF for 18 h and/or stimulated by 500 ng/mL LPS for 4 h. The level of TNF-\(\alpha\) in the culture supernatants was measured by ELISA. (**E**) THP-1 cells were pretreated with 200 MSF ug/mL or K8 lysates for 18 h and then re-stimulated with 500 ng/mL LPS for 4 h. The protein level of A20 was examined by western blot. Statistical analyses were conducted with an unpaired two-tailed \(t\)-test. NS, not significant; \({}^{**}p<0.01\); \({}^{***}p<0.001\).  
received an LPS injection (Figure 6C). All mice pre-injected with K8 lysates died 3 days after LPS injection, while about 38% of MSF-pre-injected mice survived after LPS injection until the end of the experiment (Figure 6D), suggesting that the MSF-mediated survival rate is superior to that of K8 lysates.  
In addition, the group subjected to pre-injection with MSF showed decreased serum TNF-\(\alpha\) by 25% and IL-1\(\beta\) by 30% compared with the mice only injected with LPS. The group subjected to pre-injection with MSF showed decreased serum TNF-\(\alpha\) by 15% and IL-1\(\beta\) by 6% compared with mice injected with K8 lysates. The group subjected to i.p. pre-injection with _F. glaberrima_ showed similar responses to mice only injected with LPS (Figure 7A,B). The LPS group had higher serum AST and ALT levels than the group without LPS. However, pre-injection with MSF reduced AST by 38% and ALT by 60%. In addition, the MSF group had lower serum AST and ALT levels than the K8 lysates group, by 14% and 22%, respectively. The _F. glaberrima_ group showed similar responses to mice only injected with LPS (Figure 7C,D). Mice injected with only MSF showed no significant changes in cytokine, AST, and ALT levels compared to those with no MSF.  
Figure 6: MSF decreased LPS-induced mouse mortality. (**A**) BALB/c mice (7 weeks old, 8 mice/group) were intraperitoneally (i.p.) injected with LPS at the indicated concentration. (**B**) Mice (n = 8) were i.p. injected with MSF at the indicated concentration. (**C**) Mice (n = 8) were i.p. pre-injected with MSF at the indicated concentration for 24 h and then re-injected with 20 mg/kg LPS. (**D**) Mice (n = 8) were i.p. pre-injected with PBS, 400 ug/mouse MSF, 400 ug/mouse K8 lysates, or 5 ug/mouse _F. glaberrima_ extracts for 24 h and then re-injected with 20 mg/kg LPS. Mouse mortality was observed for 5 days. \(p\) values were derived by the log-rank (Mantel–Cox) test using GraphPad Prism 5 software.