Notoginsenoside R1 improves intestinal microvascular functioning in sepsis by targeting Drp1-mediated mitochondrial quality imbalance

Abstract

Context: Sepsis can result in critical organ failure, and notoginsenoside R1 (NGR1) offers mitochondrial protection.

Objective: To determine whether NGR1 improves organ function and prognosis after sepsis by protecting mitochondrial quality.

Materials and methods: A sepsis model was established in C57BL/6 mice using cecum ligation puncture (CLP) and an in vitro model with lipopolysaccharide (LPS, 10 µg/mL)-stimulated primary intestinal microvascular endothelial cells (IMVECs) and then determine NGR1's safe dosage. Groups for each model were: in vivo-a control group, a CLP-induced sepsis group, and a CLP + NGR1 treatment group (30 mg/kg/d for 3 d); in vitro-a control group, a LPS-induced sepsis group, and a LPS + NGR1 treatment group (4 μM for 30 min). NGR1's effects on survival, intestinal function, mitochondrial quality, and mitochondrial dynamic-related protein (Drp1) were evaluated.

Results: Sepsis resulted in approximately 60% mortality within 7 days post-CLP, with significant reductions in intestinal microvascular perfusion and increases in vascular leakage. Severe mitochondrial quality imbalance was observed in IMVECs. NGR1 (IC₅₀ is 854.1 μM at 30 min) targeted Drp1, inhibiting mitochondrial translocation, preventing mitochondrial fragmentation and restoring IMVEC morphology and function, thus protecting against intestinal barrier dysfunction, vascular permeability, microcirculatory flow, and improving sepsis prognosis.

Discussion and conclusions: Drp1-mediated mitochondrial quality imbalance is a potential therapeutic target for sepsis. Small molecule natural drugs like NGR1 targeting Drp1 may offer new directions for organ protection following sepsis. Future research should focus on clinical trials to evaluate NGR1's efficacy across various patient populations, potentially leading to novel treatments for sepsis.

Keywords: Drp1; Traditional Chinese medicine; mitochondria; sepsis.

Figure 1.

Structural information and safety assessment of NGR1. (A) Structural information of NGR1. (B-F) Different parameters of animal after receiving an intraperitoneal injection of NGR1 (30 mg/kg, once per day for 3 d) after 7 d. B: Body weight change rate (7 d). C: Liver weight (% of total body weight). D: Serum ALT levels. E: Serum AST levels. F: Food intake (g/5 mice after 7 d). (G,H) The cell viability of IMVECs treated with different concentrations of NGR1 for 30 min through different assays.G: MTT asay, H:CCK-8 assay. NS, p > 0.05 compared with the control group.

Figure 2.

Effects of NGR1 on survival and intestinal function of septic mice. (A) Survival rate and survival time (n = 15). (B) HE staining of intestinal villi to observe gross morphology (400×). (C) Speckle tomography images of mesenteric perfusion and related statistics. (D) FITC-BSA leakage of mesenteric microveins (n = 8). Data are presented as mean ± standard deviation. a, p < 0.05 compared with the control group; b, p < 0.05 compared with the CLP group.

Figure 3.

Effects of NGR1 on mitochondrial quality in LPS-induced IMVECs. (A) Representative confocal images of IMVEC mitochondrial morphology in each group (bar = 25 μm) and analysis of mitochondrial skeletons using Image J software. (B) Representative confocal images of mitochondrial membrane potential (ΔΨm) of IMVECs, which were labeled with JC-1 monomer (green fluorescent probe) and JC-1 aggregate (red fluorescent probe) (bar = 25 μm). (C) Representative confocal images of ROS fluorescence intensity in IMVECs (bar = 50 μm). (D) Statistical analysis of ΔΨm and ROS in IMVECs (n = 5). Data are presented as mean ± standard deviation. a, p < 0.05 compared with the control group; b, p < 0.05 compared with the LPS group.

Figure 4.

Targeting effect of NGR1 on Drp1, verified using small-molecule microarray chips. (A) Flow chart of small-molecule microarray chips used for screening interactions with Drp1. (B) Display of chip results. From left to right are the chip scan of experimental sample Drp1-biotin and global chip scan of control sample 6× His-tag biotin; chip results are analysed in the table below. (C) From left to right, the structural formula of NGR1, overall structural diagram of NGR1 docking to Drp1 (yellow portion is Drp1, pink portion is small molecule NGR1), and local diagram of the specific interaction sites.

Figure 5.

Effects of NGR1 on Drp1 expression in subcellular fractions of IMVECs. Western blotting results showing the effect of NGR1 treatment on the expression of (A) total Drp1, (B) mitochondrial Drp1, and (C) cytoplasmic Drp1 in IMVECs under sepsis (n = 5). Data are presented as mean ± standard deviation. a, p < 0.05 compared with the control group, b, p < 0.05 compared with the LPS group. NS, p > 0.05 compared with the control group.