Inhibition of HMGB1 improves necrotizing enterocolitis by inhibiting NLRP3 via TLR4 and NF-κB signaling pathways.
To explore the relationship between high-mobility group box 1 (HMGB1) and NLR pyrin domain containing 3 (NLRP3) in the development of necrotizing enterocolitis (NEC).
NEC rat models were constructed and treated with HMGB1 inhibitor glycyrrhizin (GL) with different concentration. An inflammatory condition of intestinal tissue in newborn NEC rats was observed by hematoxylin and eosin staining. The messenger RNA (mRNA) and protein expression of HMGB1, NLRP3, toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), and caspase 1 were determined by real-time polymerase chain reaction and western blot analysis, respectively. The content of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) was determined by enzyme-linked immunosorbent assay. Human intestinal epithelial cell lines were induced to NEC by lipopolysaccharides (LPSs). LPS-induced cells were transfected with small interfering RNA-HMGB1 and NLRP3 plasmid vector. The mRNA and protein expression of HMGB1, NLRP3, TLR4, NF-κB, caspase 1, IL-1β, and TNF-α were determined by real-time PCR and western blot analysis, respectively.
The mRNA and protein expression of HMGB1 and NLRP3 in the NEC group was significantly higher than the control group. Inhibition of HMGB1 expression improved intestinal inflammation in newborn NEC rats. The expression of HMGB1, NLRP3, TLR4, NF-κB, and caspase 1 was upregulated in NEC and was weakened after treating with GL. LPS induction to intestinal epithelial cells markedly increased the expression of HMGB1, NLRP3, TLR4, NF-κB, caspase 1, IL-1β, and TNF-α. The knockdown of HMGB1 abolished the increase of expression, whereas further transfection with NLRP3 plasmid vector recovered the increase.
HMGB1 and NLRP3 were all upregulated in the development of NEC. Inhibition on HMGB1 could improve the intestinal inflammation in NEC by inhibiting NLRP3 via TLR4 and NF-κB signaling pathways.
Yu R
，Jiang S
，Tao Y
，Li P
，Yin J
，Zhou Q
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Overexpressed FOXO3 improves inflammatory status in mice by affecting NLRP3-mediated cell coronation in necrotizing colitis mice.
To investigate the relationship between FOXO3 overexpression and NLRP3 and explore the effect of FOXO3 on necrotizing colitis.
100 clean grade newborn SD (Sprague Dawley) rats were randomly divided into 4 groups: NEC group, NEC + FOXO3a group, NEC + NC group and control group. NEC rat model was established by hypoxia + hypothermia stimulation; HE staining was used for detection of the inflammation of intestinal tissue. The histological scores of intestinal tissues were histologically scored, generally, there were three types of inflammatory scoring systems including anatomically based systems, severity-based systems and quality of life systems (Lim et al., 2015) and in this study we utilized severity-based systems by HE staining. Human intestinal epithelial cell line was transfected with recombinant plasmid overexpressing FOXO3a and recombinant plasmid overexpressing NLRP3, and divided into control group, LPS group, LPS + NC group, LPS + FOXO3a group and LPS + FOXO3a + NLRP3 group; Caspase-1 was used for the detection of pyroptosis. The expressions of FOXO3a, NLRP3, cleaved Caspase-1 and the expression of TLR4 in TLR4 signaling pathway were detected by RT-qPCR and WB. IL-1β, IL-6, IL-18 and TNF-α were detected by ELISA.
(1) FOXO3a is under-expressed and NLRP3 is highly expressed in NEC neonatal rat intestinal tissue. (2) The inflammatory condition of intestinal tissue in NEC + FOXO3a group was improved compared with NEC group (P < 0.05). (3) FOXO3a was highly expressed in NEC + FOXO3a group. The expression of IL-1β, IL-6, IL-18, SOD and MDA in NEC + FOXO3a group was lower than that in NEC group. (4) The expression of IL-1β, IL-6, IL-18, SOD and MDA in intestinal epithelial cells of LPS + FOXO3a group was lower than other groups. (5) Overexpression of FOXO3a inhibits LPS-induced pyroptotic cell death in intestinal epithelial cells by inhibiting NLRP3.
Overexpression of FOXO3 in mice with necrotizing colitis can improve inflammatory conditions in mice by affecting NLRP3-mediated cell caking.
Yin Y
，Wang J
，Zhao X
，Wu X
，Zou H
，Qin Z
，Cao J
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The protective effect of formononetin on cognitive impairment in streptozotocin (STZ)-induced diabetic mice.
The present study was aimed to elucidate the pharmacological effect of Formononetin (FMN) treatment on STZ-induced diabetic cognitive dysfunction. The diabetic model was induced by an intraperitoneally injection of 180 mg/kg STZ. The animals were randomly divided into five groups: control group, streptozocin (STZ, 180 mg/kg) group, STZ + metformin (Met, 200 mg/kg) group, STZ + FMN (25 mg/kg) group, STZ + FMN (50 mg/kg) group. The mice were intragastrically administrated with metformin (Met, 200 mg/kg) or FMN (25, 50 mg/kg) once daily for 6 weeks. The blood glucose content and body weight were examined. Morris water maze test and Y maze test were used to evaluate the learning and memory abilities. The cognitive decline was reversed by regulating superoxide dismutase (SOD), malondialdehyde (MDA), tumor necrosis factor-a (TNF-α), interleukin(IL)-1β, IL-6 in serum and hippocampus. The protein expressions of high mobility group box-1 protein (HMGB1), toll like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), inhibitor of NF-κB (IκBα), p-IκBα, nuclear factor kappa-B(NF-κB), p-NF-κB, NOD-like receptor 3(NLRP3), apoptosis-associated speck-like protein containing CARD(ASC) and caspase-1 were detected. Furthermore, the SH-SY5Y cells were exposed to high glucose stimulation, FMN (2.5, 5 and 10 μM) treatment, and glycyrrhizin, the selective inhibitor of HMGB1. After an incubation for 22 h, the SH-SY5Y cells were harvested for detection. As a result, FMN treatment effectively attenuated the body weight, learning and memory abilities, as well as the levels of blood glucose, SOD, MDA, TNF-α, IL-1β, IL-6. FMN administration also downregulated the protein expressions of HMGB1, TLR4, MyD88, p-IκB, p-NF-κB, NLRP3, ASC and caspase-1. The inhibition of HMGB1 by glycyrrhizin also confirmed the involvement of HMGB1/TLR4/NF-κB/NLRP3 pathway in high glucose-induced SH-SY5Y cells. In summary, the results suggested that FMN exhibited the protective effect on STZ-induced cognitive impairment possibly via the mediation of HMGB1/TLR4/NF-κB signaling and NLRP3 inflammasome.
Wang J
，Wang L
，Zhou J
，Qin A
，Chen Z
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ResearchGate
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