3.1 Simvastatin attenuates LPS-induced systemic inflammatory responses and organ failure and extends survival
Plasma pro-inflammatory and anti-inflammatory factor levels, including IL-1β, IL-6, and TNF-α, MCP-1, IFN-γ, IL-18 and IL-10, increased significantly in the LPS group. Simvastatin pretreatment decreased plasma pro-inflammatory cytokine levels and increased IL-10 levels compared with LPS alone (Fig. 1-A). This phenomenon was verified in lung tissue at the mRNA level (Fig. 1-B). The LPS group had a large number of infiltrating granulocytes in the lungs, which was significantly improved in the simvastatin-pretreated group (Fig. 1-C).
Liver and kidney function was assessed to assess organ damage. Mice treated with simvastatin had significantly reduced serum ALT (P < 0.05), AST (P < 0.01) and CR (P < 0.01) levels compared with levels in the LPS group (Fig. 1-D). However, there was no significant difference in BUN between the two groups. Previous studies showed that a high dose of statins can cause abnormal liver function, but no significant difference in organ damage was detected between mice treated with simvastatin alone for 14 days (SIM group) and control mice (Fig. 1-D).
No deaths occurred in the control group. The survival curves for mice in the LPS group sharply declined, and all mice died within 20 h. However, in the simvastatin-treated group, the survival rate increased by 20% at 48 h and 13.3% at 72 h (P < 0.001, Fig. 2). Continued observations revealed that the two surviving mice gradually recovered their appetite and activity from day 6.
3.2 Simvastatin improves blood coagulation disorders and fibrin deposition
In this study, prophylactic administration of simvastatin helped shorten the prolongation of PT and APTT. The PLT, fibrinogen levels and ATIII dropped sharply at 12 h after the injection of endotoxin, and these decreases were significantly alleviated in the simvastatin group. After endotoxin injection, the PLT was 205 ± 28×109/L in the LPS group and 305 ± 44×109/L in the LPS+SIM group, the corresponding fibrinogen levels were 1.6 ± 0.2 g/L and 2.5 ± 0.3 g/L (P<0.01), and the corresponding ATIII levels were 44 ± 2.5% and 54± 1.6%. PAI-1 increased significantly after endotoxin administration (161±25 ng/mL), but this increase was attenuated in the simvastatin group (95±6.4 ng/mL). In addition, plasma TAT and TM showed a slight decrease in the LPS+SIM group compared to the LPS group, but the differences were not significant. FDP levels were not significantly different between the two groups (Fig. 3-A).
Histologic studies showed elevated TF levels in lung tissue in the LPS group compared with the LPS+SIM group (Fig. 3-B). Additionally, more extensive fibrin deposition was observed in various organs at 12 h after endotoxin administration, and fibrin deposition was most pronounced in small and mid-sized vessels or vasculature. Notably, simvastatin treatment significantly inhibited thrombus deposition in those organs (Fig. 3-C). These observations clearly suggest that simvastatin pretreatment during endotoxin exposure contributes to prevention of DIC development.
3.3 Simvastatin attenuates intestinal damage and apoptosis
As shown in Figure 4-A, the intestinal mucosa of mice treated with saline was intact, with well-ordered villi. However, in the LPS group, the intestinal villi were disrupted, atrophic or even ruptured at locations of necrotic epithelial cells, and the mucosa was oedematous. The damage was significantly alleviated in the simvastatin-pretreated group; although the small intestinal villi were swollen and deformed, there was no fracture in the villi barrier (Fig. 4-A).
Decreased gut epithelial apoptosis is associated with an improved survival rate in sepsis . In our study, simvastatin reversed the LPS-induced upregulation of caspase3 and downregulation of Bcl-2 at the protein level, and these results were verified at the mRNA level (Fig. 4-B, C).
3.4 Simvastatin-treated mice showed different gut microbiota compositions
The 16S rDNA sequence was used to explore the microbiota composition in the three groups. The ANOSIM of PCoA matrix scores indicated a significant separation in the microbiota composition in the three groups (P<0.05; Fig. 5-A). LPS treatment resulted in an increase in community diversity, as shown by the Shannon index, and the statin decreased community diversity, but there were no significant differences among groups (Fig. 5-C). The gut microbiota compositions at the phylum level are shown in Figure 5-B. In both of the treated groups, the top two phyla were Firmicutes and Bacteroidetes. Compared to the control group, the LPS group showed an increased abundance of Firmicutes (65.6% vs. 45.5%) and Proteobacteria (10.6% vs. 2.8%) and a decreased abundance of Bacteroidetes (20.5% vs. 45.7%). In contrast, the simvastatin-treated group showed decreased Firmicutes (60.5%) and Proteobacteria (3.5%) and increased Bacteroidetes (31.7%) compared to the LPS group and was more similar to the control group. These data suggest that the simvastatin-treated mice had a different gut microbiota composition than the LPS-treated mice but a composition similar to the control mice (Fig. 5-B).
Distinctive gut microbiota compositions in the indicated groups were also identified by linear discriminant analysis. Endotoxin treatment induced a marked increase in the abundance of harmful bacteria, including Desulfovibrio (P<0.05) in the Proteobacteria phylum and Mucispirillum (P<0.05) in the Deferribacteres phylum. Desulfovibrio can produce hydrogen sulfide to destroy intestinal epithelial cells, and Mucispirillum can directly decompose mucus [26, 27]. LPS also increased the abundance of Ruminococcaceae (Firmicutes phylum). Conversely, statin therapy triggered a considerable enrichment of bacteria in the class Bacilli; however, this expansion was not class-wide but rather was due to the expansion of Lactobacillaceae (Fig. 5-D, E).
3.5 Simvastatin alters intestinal barrier function and gut permeability
In our study, the mRNA levels of ZO-1 and JAM did not change significantly in the LPS group but were markedly increased by simvastatin treatment (Fig. 6-A). However, occludin and claudin-4 levels were sharply decreased in both the LPS and LPS+SIM groups. In Caco-2 cells, ZO-1, JAM-A, occludin and claudin-4 levels increased in a concentration-dependent manner in the simvastatin-treated group (Fig. 6-B), which was inconsistent with the in vivo experiments. We hypothesize that the intestine is a more complex system than Caco-2 cells, which can regulate TJ proteins through complex mechanisms. However, these results confirmed that statins alter the expression of TJ proteins.
Chemical barriers, including lysozyme, digestive enzymes, mucopolysaccharide and antimicrobial peptides, are also important components of the intestinal mucosal barrier. As expected, Reg3b, Defb-1 and angiogerin-1 were significantly downregulated at 12 h of endotoxin treatment, but these effects were significantly reversed in the simvastatin pretreatment group. However, Reg3 g increased 4-fold in both the LPS and LPS+SIM group (Fig. 6-C).
Mucus is another barrier that can protect epithelial cells from digestive enzymes . Core 3β1,3-N-acetylglucosaminyltransferase (C3 gnt) is responsible for glycosylation of intestinal mucins, and muc2 can disassociate pathogenic and commensal bacteria [28, 29]. As expected, the mRNA levels of muc2 and C3 gnt were significantly decreased in the LPS group but increased in the simvastatin group. Additionally, the expression levels of intestinal alkaline phosphatase (IAP, detoxifies bacterial LPS) were increased in the simvastatin treatment group. However, no significant changes in the levels of lysozyme (protects against bacterial infection) were observed (Fig. 6-D).
Intestinal inflammation is also a factor affecting intestinal permeability. The mRNA levels of inflammatory cytokines in the intestine were detected. Unexpectedly, simvastatin administration did not decrease the LPS-induced increases in IL-1β, MCP-1 and IL-6 mRNA levels. TNF-α mRNA levels were significantly upregulated and the anti-inflammatory cytokine IL-10 was downregulated by simvastatin administration compared with LPS alone (Fig. 6-E). This was inconsistent with the regulatory effect of simvastatin in the lung tissue (Fig. 1-B), which may be due to the reduction in intestinal permeability caused by simvastatin, resulting in accumulation of intestinal toxins, which in turn activates the inflammatory response.
As shown in Figure 6-F, intestinal permeability was measured at 0 h, 6 h and 12 h after LPS injection. There was no difference in intestinal permeability between the LPS group and LPS+SIM group at baseline; at 6 h, the permeability was significantly lower in the LPS+SIM group than in the LPS group, but this difference was not obvious at 12 h.
3-6 Simvastatin inhibits translocation of intestinal flora
To further explore whether simvastatin pretreatment reduced bacterial translocation, we adopted bacterial cultures in organs and blood. We found that the positive rate of bacterial culture in the LPS group was higher than that in the LPS + SIM group, especially in the liver, kidney and blood (Fig. 7-A). We further counted the number of positive bacterial colonies and found that in the liver, kidney, lung, spleen and blood, the content of translocation bacteria in the LPS group was much higher than that in the LPS + SIM group (Fig. 7, B1-5). However, no difference was found in colony units between MLNs and FLF, which are outposts for bacterial translocation (Fig. 7, B6-7).
In addition, 16 sDNA sequencing revealed that the lung tissue microbial amplification success rate reached 100% in the LPS group, higher than that in the LPS + SIM group (62.5%) and consistent with the bacterial culture results (Fig. 7-C). The LPS and LPS + SIM groups showed different distributions of translocated microbes in the lung tissues, with species and number being more abundant in the LPS group (Fig. 7-D, E, F). The significantly increased flora in the LPS group included Escherichia-Shigella, Helicobacter, Faecalibacterium, Lachnospiraceae, Bacteroides and Bifidobacterium, while Lactococcus and Streptococcus were higher in the LPS+SIM group (Fig. 7-G, H).