RESUMO
OBJECTIVE: To investigate whether Radix Sanguisorbae (RS, Diyu) could restore intestinal barrier function following sepsis using a cecal ligation and puncture (CLP)-induced septic rat model and lipopolysaccharide (LPS)-challenged IEC-6 cell model, respectively. METHODS: Totally 224 rats were divided into 4 groups including a control, sham, CLP and RS group according to a random number table. The rats in the control group were administrated with Ringer's lactate solution (30 mL/kg) with additional dopamine [10 µ g/(kg·min)] and given intramuscular injections of cefuroxime sodium (10 mg/kg) 12 h following CLP. The rats in the RS group were administrated with RS (10 mg/kg) through tail vein 1 h before CLP and treated with RS (10 mg/kg) 12 h following CLP. The rats in the sham group were only performed abdominal surgery without CLP. The rats in the CLP group were performed with CLP without any treatment. The other steps were same as control group. The effects of RS on intestinal barrier function, mesenteric microvessels barrier function, multi-organ function indicators, inflammatory response and 72 h survival window following sepsis were observed. In vitro, the effects of RS on LPS-challenged IEC-6 cell viability, the expressions of zona occludens-1 (ZO-1) and ferroptosis index were evaluated by cell counting kit-8, immunofluorescence and Western blot analysis. Bioinformatic tools were applied to investigate the pharmacological network of RS in sepsis to predict the active compounds and potential protein targets and pathways. RESULTS: The sepsis caused severe intestinal barrier dysfunction, multi-organ injury, lipid peroxidation accumulation, and ferroptosis in vivo. RS treatment significantly prolonged the survival time to 56 h and increased 72-h survival rate to 7/16 (43.75%). RS also improved intestinal barrier function and relieved intestinal inflammation. Moreover, RS significantly decreased lipid peroxidation and inhibited ferroptosis (P<0.05 or P<0.01). Administration of RS significantly worked better than Ringer's solution used alone. Using network pharmacology prediction, we found that ferroptosis and hypoxia inducible factor-1 (HIF-1 α) signaling pathways might be involved in RS effects on sepsis. Subsequent Western blot, ferrous iron measurements, and FerroOrange fluorescence of ferrous iron verified the network pharmacology predictions. CONCLUSION: RS improved the intestinal barrier function and alleviated intestinal injury by inhibiting ferroptosis, which was related in part to HIF-1 α/heme oxygenase-1/Fe2+ axis.
RESUMO
BACKGROUND: Vascular hyporeactivity and leakage are key pathophysiologic features that produce multi-organ damage upon sepsis. We hypothesized that pericytes, a group of pluripotent cells that maintain vascular integrity and tension, are protective against sepsis via regulating vascular reactivity and permeability. METHODS: We conducted a series of in vivo experiments using wild-type (WT), platelet-derived growth factor receptor beta (PDGFR-ß)-Cre + mT/mG transgenic mice and Tie2-Cre + Cx43flox/flox mice to examine the relative contribution of pericytes in sepsis, either induced by cecal ligation and puncture (CLP) or lipopolysaccharide (LPS) challenge. In a separate set of experiments with Sprague-Dawley (SD) rats, pericytes were depleted using CP-673451, a selective PDGFR-ß inhibitor, at a dosage of 40 mg/(kg·d) for 7 consecutive days. Cultured pericytes, vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were used for mechanistic investigations. The effects of pericytes and pericyte-derived microvesicles (PCMVs) and candidate miRNAs on vascular reactivity and barrier function were also examined. RESULTS: CLP and LPS induced severe injury/loss of pericytes, vascular hyporeactivity and leakage (P < 0.05). Transplantation with exogenous pericytes protected vascular reactivity and barrier function via microvessel colonization (P < 0.05). Cx43 knockout in either pericytes or VECs reduced pericyte colonization in microvessels (P < 0.05). Additionally, PCMVs transferred miR-145 and miR-132 to VSMCs and VECs, respectively, exerting a protective effect on vascular reactivity and barrier function after sepsis (P < 0.05). miR-145 primarily improved the contractile response of VSMCs by activating the sphingosine kinase 2 (Sphk2)/sphingosine-1-phosphate receptor (S1PR)1/phosphorylation of myosin light chain 20 pathway, whereas miR-132 effectively improved the barrier function of VECs by activating the Sphk2/S1PR2/zonula occludens-1 and vascular endothelial-cadherin pathways. CONCLUSIONS: Pericytes are protective against sepsis through regulating vascular reactivity and barrier function. Possible mechanisms include both direct colonization of microvasculature and secretion of PCMVs.
