Sinomenine hydrochloride protects against polymicrobial sepsis via autophagy

Abstract

Sepsis, a systemic inflammatory response to infection, is the major cause of death in intensive care units (ICUs). The mortality rate of sepsis remains high even though the treatment and understanding of sepsis both continue to improve. Sinomenine (SIN) is a natural alkaloid extracted from Chinese medicinal plant Sinomenium acutum, and its hydrochloride salt (Sinomenine hydrochloride, SIN-HCl) is widely used to treat rheumatoid arthritis (RA). However, its role in sepsis remains unclear. In the present study, we investigated the role of SIN-HCl in sepsis induced by cecal ligation and puncture (CLP) in BALB/c mice and the corresponding mechanism. SIN-HCl treatment improved the survival of BALB/c mice that were subjected to CLP and reduced multiple organ dysfunction and the release of systemic inflammatory mediators. Autophagy activities were examined using Western blotting. The results showed that CLP-induced autophagy was elevated, and SIN-HCl treatment further strengthened the autophagy activity. Autophagy blocker 3-methyladenine (3-MA) was used to investigate the mechanism of SIN-HCl in vitro. Autophagy activities were determined by examining the autophagosome formation, which was shown as microtubule-associated protein light chain 3 (LC3) puncta with green immunofluorescence. SIN-HCl reduced lipopolysaccharide (LPS)-induced inflammatory cytokine release and increased autophagy in peritoneal macrophages (PM). 3-MA significantly decreased autophagosome formation induced by LPS and SIN-HCl. The decrease of inflammatory cytokines caused by SIN-HCl was partially aggravated by 3-MA treatment. Taken together, our results indicated that SIN-HCl could improve survival, reduce organ damage, and attenuate the release of inflammatory cytokines induced by CLP, at least in part through regulating autophagy activities.

Figure 1

The structure of sinomenine hydrochloride (SIN-HCl).

Figure 2

SIN-HCl protected a mouse model against polymicrobial sepsis and attenuated the multiple organ dysfunction and systemic inflammatory response. (A) SIN-HCl improved the survival rate of mice with CLP-induced sepsis. BALB/c mice were subjected to CLP surgery. SIN-HCl (100 mg/kg) was administered by hypodermic injection at 0 h (A1) or 4 h (A2) after CLP surgery. NS was used as the control. * p < 0.05 when compared with CLP + NS group. Data were pooled from three experiments, n = 10 per group in each experiment; (B) The lungs, liver, and kidneys were stained with HE. The organ damage induced by CLP was attenuated significantly in SIN-HCl-treated BALB/c mice, as evidenced by less inflammatory cell infiltration, reduced exudate blockage of capillary, and less substantial interstitial cellular degeneration and necrosis in the lungs, liver, and kidneys. Scale bar was equivalent to 50 μm. Images were the selected representatives of each group, which had six to eight mice; (C) Semi-quantitative analysis of lung, liver, and kidney injury. ** p < 0.01 when compared with sham BALB/c mice, n ≥ 6; ^# p < 0.05 when compared with the NS-treated BALB/c mice, n ≥ 6; and (D) Biochemical measurements of the serum (D1–D4) were performed on automatic biochemical analyzer, and concentrations of serum IL-6 and TNF-α (D5) were determined by ELISA. ** p < 0.01 when compared with sham BALB/c mice, n ≥ 6; ^# p < 0.05 and ^## p < 0.01 when compared with the CLP and NS-treated BALB/c mice, n ≥ 6.

Figure 2

SIN-HCl protected a mouse model against polymicrobial sepsis and attenuated the multiple organ dysfunction and systemic inflammatory response. (A) SIN-HCl improved the survival rate of mice with CLP-induced sepsis. BALB/c mice were subjected to CLP surgery. SIN-HCl (100 mg/kg) was administered by hypodermic injection at 0 h (A1) or 4 h (A2) after CLP surgery. NS was used as the control. * p < 0.05 when compared with CLP + NS group. Data were pooled from three experiments, n = 10 per group in each experiment; (B) The lungs, liver, and kidneys were stained with HE. The organ damage induced by CLP was attenuated significantly in SIN-HCl-treated BALB/c mice, as evidenced by less inflammatory cell infiltration, reduced exudate blockage of capillary, and less substantial interstitial cellular degeneration and necrosis in the lungs, liver, and kidneys. Scale bar was equivalent to 50 μm. Images were the selected representatives of each group, which had six to eight mice; (C) Semi-quantitative analysis of lung, liver, and kidney injury. ** p < 0.01 when compared with sham BALB/c mice, n ≥ 6; ^# p < 0.05 when compared with the NS-treated BALB/c mice, n ≥ 6; and (D) Biochemical measurements of the serum (D1–D4) were performed on automatic biochemical analyzer, and concentrations of serum IL-6 and TNF-α (D5) were determined by ELISA. ** p < 0.01 when compared with sham BALB/c mice, n ≥ 6; ^# p < 0.05 and ^## p < 0.01 when compared with the CLP and NS-treated BALB/c mice, n ≥ 6.

Figure 3

CLP-induced autophagy in multiple organs was strengthened in SIN-HCl-treated mice. (A) SIN-HCl treatment strengthened CLP-induced autophagy in multiple organs of the mice. The protein level of LC3 was measured using an anti-LC3 antibody. The ratios of LC3-II:LC3-I in the lungs, liver, and kidneys were higher at 12 h after CLP procedure or SIN-HCl treatment. SIN-HCl further increased the ratios of LC3-II:LC3-I in the lungs and livers of mice that had undergone the CLP; (B) Western blotting was quantitatively analyzed, * p < 0.05 when compared with sham BALB/c mice, n ≥ 5; ^# p < 0.05 when compared with the CLP + NS-treated BALB/c mice, n ≥ 5.

Figure 4

SIN-HCl blocked LPS-induced inflammatory cytokine release and increased autophagy in peritoneal macrophages (PM). (A) Primary PM were cultured for 12 h with LPS (100 ng/mL), SIN-HCl (100 mM), or a combination of these two reagents (as indicated). The levels of IL-6 and TNF-α in culture media were measured. ** p < 0.01 when compared with PBS group, n ≥ 6; ^# p < 0.05 when compared with the LPS group, n ≥ 6; (B) Primary PM were treated with LPS (100 ng/mL), SIN-HCl (100 mM), or a combination of these two reagents (as indicated) for 12 h before immunofluorescent detection of LC3 protein using an anti-LC3 antibody, Scale bar = 10 μm and (C) Autophagosomes per cell in PM were counted in at least 30 cells. ** p < 0.01 when compared with PBS group, n ≥ 4; ^# p < 0.05 when compared with the LPS group, n ≥ 4.

Figure 5

The effects of 3-MA on SIN-HCl-induced autophagy and inflammatory responses in PM. (A) Primary PM were cultured for 12 h with 3-MA (5 mM), LPS (100 ng/mL), SIN-HCl (100 mM), or a combination of these two reagents (as indicated). The levels of IL-6 and TNF-α in culture media were measured. ** p < 0.01 when compared with PBS group, n ≥ 6; ^# p < 0.05 when compared with the LPS group, n ≥ 6; (B) Primary PM were treated with 3-MA (5 mM), LPS (100 ng/mL), SIN-HCl (100 mM), or a combination of these reagents (as indicated) for 12 h before immunofluorescent detection of LC3 protein using an anti-LC3 antibody. Scale bar = 10 μm; (C) Autophagosomes in at least 30 PM cells were counted. Scale bar was equivalent to 10 μm. ** p < 0.01 when compared with PBS group, n ≥ 4; ^# p < 0.05 when compared with the LPS group, n ≥ 4.