To investigate the effects of paeoniflorin (PF) on lipopolysaccharide (LPS) - induced aerobic glycolysis in renal tubular epithelial cell line HK-2 and its underlying mechanism of action.

This study consists of a preliminary experiment and a formal experiment. Preliminary experiment: CCK-8 assay and RT-qPCR were used respectively to measure cell viability and mRNA expression levels of inflammatory factors in HK-2 cells after LPS stimulation to determine the optimal LPS concentration for modeling as well as to evaluate the toxicity of PF and screen for its appropriate concentration. Formal experiment: HK-2 cells were divided into control group (CON group), LPS group, LPS+PF group and LPS+PF+ 740Y-P group. LPS was used to establish a cell model of sepsis associated-acute kidney injury (SA-AKI) in HK-2 cells, and then the cell model was treated with PF and PI3K activator 740Y-P, correspondingly for 24 h. CCK-8 assay was employed to detect cell viability, and Extracellular Acidification Rate (ECAR) Kit was utilized to measure the rate. The contents of IL-1β, IL-18, lactic acid (Lac) and lactate dehydrogenase A (LDHA) were determined with ELISA. Western blotting was applied to detect the expression of p-PI3K, p-AKT, HIF-1α, pyruvate kinase 2 (PKM2, a key enzyme in aerobic glycolysis) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3), and immunofluorescence assay was performed to observe the expression and distribution of PKM2.

① Our preliminary experiment identified that the optimal concentration of LPS for modeling was 20. 0 μg/mL, a safe dosage range of PF was 0~100. 0 μmol/L, and its optimal therapeutic concentration was 25. 0 μmol/L. ② Compared with the CON group, LPS stimulation resulted in significantly decreased cell viability (P<0. 05), increased ECAR (P<0. 05), elevated contents of IL-1β, IL-18, Lac and LDHA (P<0. 05), up-regulated protein levels of p-PI3K, p-AKT, HIF-1α, p-PKM2 and NLRP3 (P< 0. 05), and enhanced fluorescence intensity of PKM2 in the nucleus of cells (P<0. 05). Compared with the model group, PF treatment reversed all above effects induced by LPS stimulation (all P<0. 05). Compared with the LPS+PF group, in the LPS+PF+740Y-P group, ECAR was elevated (P<0. 05), the contents of IL-1β, IL-18, Lac and LDHA were increased (P<0. 05), and the relative expression levels of p-PI3K, p-AKT, HIF-1α, p-PKM2 and NLRP3 were increased (P<0. 05), and the fluorescence intensity of PKM2 was strengthened (P<0. 05) and enhanced in the nucleus (P<0. 05).

PF reduces aerobic glycolysis in HK-2 cells and alleviates the inflammatory response by inhibiting the PI3K/AKT/HIF-1α signaling pathway.

To investigate the effects of cannabinoid receptor agonist, WIN55212-2 (WIN), on acute kidney injury (AKI) in mice with sepsis and its underlying mechanism.

Twenty-four healthy male mice were divided into (n=6): Control group, sepsis group (LPS group), sepsis+WIN55212-2 group (LPS+WIN group) and sepsis+WIN55212-2+mTOR activator MHY1485 group (LPS+WIN+MHY group). The sepsis model was constructed by intraperitoneal injection of LPS. After the tissue and blood samples were harvested in 24 h after modeling, ELISA was used to determine the contents of IL-1β, IL-18 and lactate dehydrogenase A (LDHA) in the renal tissues, as well as Scr, kidney injury molecule-1 (KIM-1) and lactic acid (LA) in the serum. HE staining was employed to observe the pathological changes of kidney tissue and Paller score was calculated. The expression of p-AKT, p-mTOR and 6-phosphofructokinase-2/fructose-2, 6-biphosphatase 3 (PFKFB3) in the renal tissues was detected by Western blotting.

Compared with the Control group, the LPS group had obvious kidney tissue damage, increased Paller score (P < 0.05), increased serum contents of lactic acid, Scr and KIM-1 (P < 0.05), up-regulated contents of LDHA, IL-1β and IL-18 in the renal tissues (P < 0.05), and elevated expression levels of p-AKT, p-mTOR and PFKFB3 in the renal tissues (P < 0.05). Milder pathological injury in kidney tissue, decreased Paller score (P < 0.05), reduced contents of lactic acid, Scr and KIM-1 (P < 0.05), decreased contents of LDHA, IL-1β and IL-18 (P < 0.05), and lower expression of p-AKT, p-mTOR and PFKFB3 were observed in the LPS+WIN group than the LPS group (P < 0.05). The LPS+WIN+MHY group had notably higher Paller score (P < 0.05), raised contents of lactic acid, Scr and KIM-1 (P < 0.05), increased contents of LDHA, IL-1β and IL-18 (P < 0.05), and up-regulated expression of p-AKT, p-mTOR and PFKFB3 (P < 0.05) when compared with the LPS+WIN group.

WIN can alleviate AKI in sepsis, and it may reduce the level of glycolysis in renal tissue, and alleviate inflammation through inhibiting AKT/mTOR/PFKFB3 signaling pathway.

To investigate the therapeutic efficacy and underlying mechanism of naringin (Ng) for intestinal mucosal injury in mice with intra-abdominal infection (IAI).

A mouse IAI model was established by intraperitoneal injection (i.p.) of lipopolysaccharide (LPS). Sixty-four healthy male mice were randomly assigned to 4 groups (n=16): Control group (0.2 mL PBS), LPS group (LPS 10 mg/kg), Ng group (50 mg/kg Ng+10 mg/kg LPS), and BzATP group (5 mg/kg BzATP +50 mg/kg Ng+10 mg/kg LPS). Ng and BzATP were administered i.p. 1 h before modeling, while the Control group received an equivalent volume of PBS. At 24 h post-modeling, 6 mice from each group were randomly selected and sacrificed for ileum collection, and the histopathological changes in the ileal mucosa and Chiu's score were assessed using HE staining. Immunofluorescence assay was employed to investigate the expression and distribution of intestinal mucosal P2X7, pyroptosis effector protein GSDMD, and macrophage marker CD68. Western blotting was conducted to detect the expression of P2X7 and pyroptosis-related proteins NLRP3, Caspase11, GSDMD, and GSDMD-N protein in ileum tissue. ELISA was utilized to measure the contents of inflammatory factors IL-1β, IL-18, and IL-10 in the ileum. The mouse sepsis score (MSS) was recorded within 7 d.

In comparison to the Control group, the LPS group exhibited significantly higher MSS and Chiu's scores (P < 0.05), along with inflammation-associated damage to the ileal mucosa; Immunofluorescence assay displayed consistent and intensified distribution of intestinal mucosal P2X7, GSDMD, and CD68 (P < 0.05); Levels of pro-inflammatory factors IL-1β and IL-18 were increased, while that of anti-inflammatory factor IL-10 was decreased (P < 0.05); Additionally, the protein expression of P2X7, NLRP3, Caspase11, GSDMD, and GSDMD-N in ileum tissue was increased (P < 0.05). In contrast to the LPS group, the Ng group displayed a decrease in MSS; HE staining and Chiu's scores indicated a reduction in inflammatory damage to the intestinal mucosa; Moreover, fluorescence signals for P2X7, GSDMD, and CD68 were decreased (P < 0.05). Levels of IL-1β and IL-18 were decreased (P < 0.05), while that of IL-10 was elevated (P < 0.05). The expression of P2X7, NLRP3, Caspase11, GSDMD, and GSDMD-N protein was decreased (P < 0.05). The results of the BzATP group were in contrast to those of the Ng group.

Elevoted expression of P2X7 receptor and Caspase11-mediated non-canonical pyroptosis aggravates the intestinal mucosal injury IAI mice. Ng may ameliorate the injury in IAI mice by suppressing P2X7 receptor and modulating non-canonical pyroptosis.

To observe the effect of tanshinoneⅡA（TanⅡA）on acute lung injury（ALI）in septic mice, and to explore its effect on neutrophil extracellular traps（NETs）and its possible mechanism.

A mouse model of sepsis was established by intraperitoneal injection of lipopolysaccharide（LPS）at a dose of 10 mg/kg. Thirty male C57BL/6 mice aged 6~8 weeks were randomly divided into 5 groups（n=6）: Normal group（Nor group）, LPS group, LPS+TanⅡA group（30 mg/kg TanⅡA was injected intraperitoneally 30 min before LPS injection）, LPS+LY294002 group（pre-injection of 30 mg/kg LY294002）, and LPS+DMSO group（pre-injection of the same amount of DMSO）. At 12 h after modeling, the lung tissues were collected, lung coefficients were calculated, and pathological changes of lung tissue were observed with HE staining. ELISA was used to detect the levels of inflammatory factors IL-1β and IL-10 and NETs markers MPO-DNA complex and citrolinated histone H3（Cit H3）in lung tissue. Western blotting was applied to detect the phosphorylation of the PI3K/AKT signaling pathway.

Compared with the Nor group, the LPS group had an increased lung coefficient, significantly damaged lung tissue, increased levels of IL-1β（P<0.05）and decreased level of IL-10（P<0.05）, and elevated MPO-DNA complex and Cit H3 levels（P<0.05）as well as the levels of p-PI3K and p-AKT（P<0.05）. While, the above conditions were much better in the LPS+TanⅡA group and the LPS+LY294002 group than the LPS group, with decreased lung coefficient, alleviated lung injury by HE staining and Smith scores, increased level of IL-1β and decreased IL-10 level in the lung tissue（P<0.05）, reduced levels of MPO-DNA complex and Cit H3（P<0.05）and p-PI3K and p-AKT（P<0.05）.

TanⅡA can alleviate sepsis-induced ALI, and its mechanism may be through inhibiting PI3K/AKT signaling pathway, regulating the level of NETs, and thus reducing the inflammatory response.