Baicalin improves survival in a murine model of polymicrobial sepsis via suppressing inflammatory response and lymphocyte apoptosis

Abstract

Background: An imbalance between overwhelming inflammation and lymphocyte apoptosis is the main cause of high mortality in patients with sepsis. Baicalin, the main active ingredient of the Scutellaria root, exerts anti-inflammatory, anti-apoptotic, and even antibacterial properties in inflammatory and infectious diseases. However, the therapeutic effect of baicalin on polymicrobial sepsis remains unknown.

Methodology/principal findings: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Mice were infused with baicalin intraperitoneally at 1 h, 6 h and 12 h after CLP. Survival rates were assessed over the subsequent 8 days. Bacterial burdens in blood and peritoneal cavity were calculated to assess the bacterial clearance. Neutrophil count in peritoneal lavage fluid was also calculated. Injuries to the lung and liver were detected by hematoxylin and eosin staining. Levels of cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10 and IL-17, in blood and peritoneum were measured by enzyme-linked immunosorbent assay. Adaptive immune function was assessed by apoptosis of lymphocytes in the thymus and counts of different cell types in the spleen. Baicalin significantly enhanced bacterial clearance and improved survival of septic mice. The number of neutrophils in peritoneal lavage fluid was reduced by baicalin. Less neutrophil infiltration of the lung and liver in baicalin-treated mice was associated with attenuated injuries to these organs. Baicalin significantly reduced the levels of proinflammatory cytokines but increased the level of anti-inflammatory cytokine in blood and peritoneum. Apoptosis of CD3(+) T cell was inhibited in the thymus. The numbers of CD4(+), CD8(+) T lymphocytes and dendritic cells (DCs) were higher, while the number of CD4(+)CD25(+) regulatory T cells was lower in the baicalin group compared with the CLP group.

Conclusions/significance: Baicalin improves survival of mice with polymicrobial sepsis, and this may be attributed to its antibacterial property as well as its anti-inflammatory and anti-apoptotic effects.

Figure 1. Survival rate of septic mice after injection with baicalin.

Baicalin improves survival of polymicrobial sepsis in CLP mice. In the bai group, mice were treated with intra-peritoneal baicalin (100 mg/kg) at 1 h, 6 h and 12 h after CLP. Normal saline of equal volume was administered in sham and CLP groups. Bai, baicalin group; CLP, CLP group. Survival analyses with log-rank test. ** P<0.01.

Figure 2. Bacterial clearance in blood (A) and peritoneum (B) in septic mice after baicalin treatment.

Baicalin enhanced bacterial clearance in blood (A) and peritoneum (B). Blood and peritoneal lavage fluid were harvested 24 h after CLP. Bai, baicalin group; CLP, CLP group. Data analyses with one-way ANOVA and Newman-Keuls. ** P<0.01.

Figure 3. The number of neutrophils in peritoneum in septic mice after administration of baicalin.

Baicalin reduced the number of neutrophils in peritoneum. Peritoneal lavage fluid were harvested 24 h after CLP. Bai, baicalin group; CLP, CLP group. Data analyses with one-way ANOVA and Newman-Keuls. * P<0.05.

Figure 4. Histopathological changs of septic mice after baicalin treatment.

The tissues were harvested 24 h after CLP for histopathologic examination using hematoxylin and eosin staining. Representative images from six animals per group were shown. Histopathological tests showed milder impairment in lung and liver after baicalin administration. A, After baicalin treatment, lung showed less neutrophil accumulation and alveolar destruction. B, Hepatocytes were protected and hepatic sinusoid was also preserved in baicalin treated mice. C, the severity of lung injury was scored as described in Materials and methods. D, the severity of liver injury was scored as above. ** P<0.01 vs. sham. ## P<0.01 vs. CLP. Bai, baicalin group; CLP, CLP group.

Figure 5. Cytokines expression of septic mice after baicalin injection.

Baicalin reduced excretion of proinflammatory cytokines while increased anti-inflammatory cytokine. Samples were collected 24 h after CLP. Proinflammatory cytokines included TNF-alpha, IL-6 and IL-17 (A, B, D in blood and E, F, H in peritoneum). IL-10 represented the anti-inflammatory cytokine (C in blood and G in peritoneum). PLF, peritoneal lavage fluid; Bai, baicalin group; CLP, CLP group. Data analyses with one-way ANOVA and Newman-Keuls. * P<0.05, ** P<0.01.

Figure 6. Cell counts in the spleen and apoptosis in the thymus in septic mice after baicalin administration.

(A) Baicalin increased the numbers of CD4⁺ and CD8⁺ T lymphocytes, as well as CD11c⁺ dendritic cells, but not CD19⁺ B lymphocytes in spleen. (B) Baicalin reduced the number of regulative T cells in spleen. (C, D) Baicalin inhibited lymphocyte apoptosis in thymus. Bai, baicalin group; CLP, CLP group. Data analyses with one-way ANOVA and Newman-Keuls. *P<0.05, ** P<0.01.