OBJECTIVE: To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats. METHODS: Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 μmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors-transforming growth factor β ₁ (TGF-β ₁), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type Ⅱ. RESULTS: The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L ( P<0.05), so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-β ₁, IL-6, and TNF-α in cells significantly increased when compared with the control group ( P<0.05). However, intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group ( P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 ( P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type Ⅱ expression. CONCLUSION VX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.
Rats ; Animals ; Chondrocytes/metabolism* ; Matrix Metalloproteinase 13/metabolism* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha/metabolism* ; Collagen Type II/metabolism* ; Interleukin-6 ; Lipopolysaccharides/pharmacology* ; NF-E2-Related Factor 2/pharmacology* ; Inflammation/drug therapy* ; Osteoarthritis/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Dipeptides ; para-Aminobenzoates

OBJECTIVE: To examine the therapeutic effect of Fangji Fuling Decoction (FFD) on sepsis through network pharmacological analysis combined with in vitro and in vivo experiments. METHODS: A sepsis mouse model was constructed through intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS). RAW264.7 cells were stimulated by 250 ng/mL LPS to establish an in vitro cell model. Network pharmacology analysis identified the key molecular pathway associated with FFD in sepsis. Through ectopic expression and depletion experiments, the effect of FFD on multiple organ damage in septic mice, as well as on cell proliferation and apoptosis in relation to the mitogen-activated protein kinase 14/Forkhead Box O 3A (MAPK14/FOXO3A) signaling pathway, was analyzed. RESULTS: FFD reduced organ damage and inflammation in LPS-induced septic mice and suppressed LPS-induced macrophage apoptosis and inflammation in vitro (P<0.05). Network pharmacology analysis showed that FFD could regulate the MAPK14/FOXO signaling pathway during sepsis. As confirmed by in vitro cell experiments, FFD inhibited the MAPK14 signaling pathway or FOXO3A expression to relieve LPS-induced macrophage apoptosis and inflammation (P<0.05). Furthermore, FFD inhibited the MAPK14/FOXO3A signaling pathway to inhibit LPS-induced macrophage apoptosis in the lung tissue of septic mice (P<0.05). CONCLUSION FFD could ameliorate the LPS-induced inflammatory response in septic mice by inhibiting the MAPK14/FOXO3A signaling pathway.
Mice ; Animals ; Mitogen-Activated Protein Kinase 14/metabolism* ; Wolfiporia ; Lipopolysaccharides/pharmacology* ; Sepsis/complications* ; Signal Transduction ; Inflammation/drug therapy* ; Oxygen Radioisotopes

This study aimed to investigate the biological effects and underlying mechanisms of the total ginsenosides from Panax ginseng stems and leaves on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice. Sixty male C57BL/6J mice were randomly divided into a control group, a model group, the total ginsenosides from P. ginseng stems and leaves normal administration group(61.65 mg·kg~(-1)), and low-, medium-, and high-dose total ginsenosides from P. ginseng stems and leaves groups(15.412 5, 30.825, and 61.65 mg·kg~(-1)). Mice were administered for seven continuous days before modeling. Twenty-four hours after modeling, mice were sacrificed to obtain lung tissues and calculate lung wet/dry ratio. The number of inflammatory cells in bronchoalveolar lavage fluid(BALF) was detected. The levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in BALF were detected. The mRNA expression levels of IL-1β, IL-6, and TNF-α, and the levels of myeloperoxidase(MPO), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in lung tissues were determined. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in lung tissues. The gut microbiota was detected by 16S rRNA sequencing, and gas chromatography-mass spectrometry(GC-MS) was applied to detect the content of short-chain fatty acids(SCFAs) in se-rum. The results showed that the total ginsenosides from P. ginseng stems and leaves could reduce lung index, lung wet/dry ratio, and lung damage in LPS-induced ALI mice, decrease the number of inflammatory cells and levels of inflammatory factors in BALF, inhibit the mRNA expression levels of inflammatory factors and levels of MPO and MDA in lung tissues, and potentiate the activity of GSH-Px and SOD in lung tissues. Furthermore, they could also reverse the gut microbiota disorder, restore the diversity of gut microbiota, increase the relative abundance of Lachnospiraceae and Muribaculaceae, decrease the relative abundance of Prevotellaceae, and enhance the content of SCFAs(acetic acid, propionic acid, and butyric acid) in serum. This study suggested that the total ginsenosides from P. ginseng stems and leaves could improve lung edema, inflammatory response, and oxidative stress in ALI mice by regulating gut microbiota and SCFAs metabolism.
Mice ; Male ; Animals ; Ginsenosides/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6 ; Panax/genetics* ; Lipopolysaccharides/adverse effects* ; Gastrointestinal Microbiome ; RNA, Ribosomal, 16S ; Mice, Inbred C57BL ; Acute Lung Injury/genetics* ; Lung/metabolism* ; Superoxide Dismutase/metabolism* ; Plant Leaves/metabolism* ; RNA, Messenger

OBJECTIVE: To investigate and reveal the underlying mechanism of the effect of total saponins from Dioscoreae nipponica Makino (TSDN) on the arachidonic acid pathway in monosodium urate (MSU) crystal-induced M1-polarized macrophages. METHODS: M1 polarization of RAW264.7 cells were induced by 1 µ g/mL lipopolysaccharide (LPS). The methylthiazolyldiphenyl-tetrazolium bromide method was then used to screen the concentration of TSDN. MSU (500 µ g/mL) was used to induce the gouty arthritis model. Afterwards, 10 µ g/L TSDN and 8 µ mol/L celecoxib, which was used as a positive control, were added to the above LPS and MSU-induced cells for 24 h. The mRNA and protein expressions of cyclooxygenase (COX) 2, 5-lipoxygenase (5-LOX), microsomal prostaglandin E synthase derived eicosanoids (mPGES)-1, leukotriene B (LTB)4, cytochrome P450 (CYP) 4A, and prostaglandin E2 (PGE₂) were tested by real-time polymerase chain reaction and Western blotting, respectively. The enzyme-linked immunosorbent assay was used to test the contents of M1 markers, including inducible nitric oxid synthase (NOS) 2, CD80, and CD86. RESULTS: TSDN inhibited the proliferation of M1 macrophages and decreased both the mRNA and protein expressions of COX2, 5-LOX, CYP4A, LTB4, and PGE₂ (P<0.01) while increased the mRNA and protein expression of mPGES-1 (P<0.05 or P<0.01). TSDN could also significantly decrease the contents of NOS2, CD80, and CD86 (P<0.01). CONCLUSION TSDN has an anti-inflammation effect on gouty arthritis in an in vitro model by regulating arachidonic acid signaling pathway.
Uric Acid/metabolism* ; Arachidonic Acid/metabolism* ; Dioscorea ; Arthritis, Gouty ; Lipopolysaccharides ; Saponins/pharmacology* ; Macrophages ; Signal Transduction ; RNA, Messenger/metabolism*

OBJECTIVES: To study the effect of procalcitonin (PCT) on lipopolysaccharide (LPS)-induced expression of the pyroptosis-related proteins nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and caspase-1 in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were induced by LPS to establish a model of sepsis-induced inflammatory endothelial cell injury. The experiment was divided into two parts. In the first part, HUVECs were randomly divided into four groups: normal control, LPS (1 μg/mL), PCT (10 ng/mL), and LPS+PCT (n=3 each). In the second part, HUVECs were randomly grouped: normal control, LPS, and LPS+PCT of different concentrations (0.1, 1, 10, and 100 ng/mL) (n=3 each). Quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of NLRP3 and caspase-1 in each group. RESULTS: In the first experiment: compared with the normal control group, the PCT, LPS, and LPS+PCT groups had significantly upregulated mRNA and protein expression levels of NLRP3 and caspase-1 (P<0.05); compared with the LPS group, the LPS+PCT group had significantly downregulated mRNA and protein expression levels of NLRP3 and caspase-1 (P<0.05). In the second experiment: compared with those in the LPS group, the mRNA and protein expression levels of NLRP3 and caspase-1 in the LPS+PCT of different concentrations groups were significantly downregulated in a concentration-dependent manner (P<0.05). CONCLUSIONS LPS can promote the expression of the pyroptosis-related proteins NLRP3 and caspase-1 in HUVECs, while PCT can inhibit the LPS-induced expression of the pyroptosis-related proteins NLRP3 and caspase-1 in HUVECs in a concentration-dependent manner.
Humans ; Caspase 1/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Lipopolysaccharides/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Procalcitonin ; Nucleotides/pharmacology*

OBJECTIVE: To investigate the inflammatory effects of Cinobufotalin on monocytes in resting state and macrophages in activated state and its molecular mechanism. METHODS: THP-1 cells were stimulated with Phorbol 12-myristate 13-acetate to induce differentiation into macrophages. Lipopolysaccharides was added to activate macrophages in order to establish macrophage activation model. Cinobufotalin was added to the inflammatory cell model for 24 h as a treatment. CCK-8 was used to detect cell proliferation, Annexin V /PI double staining flow cytometry was used to detect cell apoptosis, flow cytometry was used to detect macrophage activation, and cytometric bead array was used to detect cytokines. Transcriptome sequencing was used to explore the gene expression profile regulated by Cinobufotalin. Changes in the significantly regulated molecules were verified by real-time quantitative polymerase chain reaction and Western blot. RESULTS: 1∶25 concentration of Cinobufotalin significantly inhibited the proliferation of resting monocytes(P<0.01), and induced apoptosis(P<0.01), especially the activated macrophages(P<0.001, P<0.001). Cinobufotalin significantly inhibited the activation of macrophages, and significantly down-regulated the inflammatory cytokines(IL-6, TNF-α, IL-1β, IL-8) released by activated macrophages(P＜0.001). Its mechanism was achieved by inhibiting TLR4/MYD88/P-IκBa signaling pathway. CONCLUSION Cinobufotalin can inhibit the inflammatory factors produced by the over-activation of macrophages through TLR4/MYD88/P-IκBa pathway, which is expected to be applied to the treatment and research of diseases related to the over-release of inflammatory factors.
Humans ; Toll-Like Receptor 4/metabolism* ; Myeloid Differentiation Factor 88/genetics* ; Macrophages/metabolism* ; Cytokines/metabolism* ; Lipopolysaccharides/pharmacology* ; NF-kappa B

Increased intestinal barrier permeability, leaky gut, has been reported in patients with autism. However, its contribution to the development of autism has not been determined. We selected dextran sulfate sodium (DSS) to disrupt and metformin to repair the intestinal barrier in BTBR T⁺tf/J autistic mice to test this hypothesis. DSS treatment resulted in a decreased affinity for social proximity; however, autistic behaviors in mice were improved after the administration of metformin. We found an increased affinity for social proximity/social memory and decreased repetitive and anxiety-related behaviors. The concentration of lipopolysaccharides in blood decreased after the administration of metformin. The expression levels of the key molecules in the toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-nuclear factor kappa B (NF-κB) pathway and their downstream inflammatory cytokines in the cerebral cortex were both repressed. Thus, "leaky gut" could be a trigger for the development of autism via activation of the lipopolysaccharide-mediated TLR4-MyD88-NF-κB pathway.
Mice ; Animals ; NF-kappa B ; Myeloid Differentiation Factor 88/metabolism* ; Lipopolysaccharides/pharmacology* ; Toll-Like Receptor 4/metabolism* ; Autistic Disorder/metabolism* ; Signal Transduction/physiology*

Objective: To investigate the effects of colony-stimulating factor 1 receptor (CSF-1R) inhibitor pexidartinib (PLX3397) on the senescence of bone marrow-derived macrophages (BMDM) stimulated by lipopolysaccharide (LPS). Methods: BMDM were isolated and cultured from femurs and tibiae of 10 male C57BL/6 mice aged 6-8 weeks (obtained from Laboratory Animal Center of Guizhou Medical University). They were divided into blank control group, LPS group (treated with 1 μg/ml LPS for 24 h) as well as low, medium and high concentration PLX3397 pretreatment groups (treated with 100, 500 and 1 000 nmol/L PLX3397 for 4 h respectively followed by 1 μg/ml LPS for 24 h). The corresponding markers of macrophages were detected by flow cytometry. Cell viability was detected by cell counting kit-8 and cellular senescence was detected by senescence-associated-β-galactosidase (SA-β-gal) staining. Meanwhile, protein expressions of cycle-dependent kinase inhibitor p16, p21 and CSF-1R were detected by Western blotting, and the expressions of p16 and p21 were detected by intracellular immunofluorescence. Real-time fluorescence quantitative PCR (RT-qPCR) was used to investigate the mRNA levels of senescence-associated secretory phenotype (SASP) genes including interleukin (IL), IL-1β, chemokine-1/10 (CXCL-1/10), matrix metalloproteinase-8 (MMP-8), and transforming growth factor-β (TGF-β). Results: The rate of SA-β-gal positive staining in medium and high concentration PLX3397 pretreatment groups [(39.33±4.93)% and (36.33±3.06)% respectively] were significantly downregulated compared with LPS group [(52.00±3.00)%] (P=0.020, P=0.005). The expression of CSF-1R protein in low, medium and high concentration PLX3397 pretreatment groups were (0.74±0.18, 0.61±0.07, 0.54±0.06), all of which were significantly lower than that in LPS group (1.16±0.08) (P=0.013, P=0.002, P<0.001). The expression levels of CSF-1R mRNA in low, medium and high concentration PLX3397 pretreatment groups (1.04±0.06, 0.90±0.05, 1.18±0.08) showed similar trend (2.90±0.25) (P<0.001). The average fluorescence intensity of p16 in all PLX3397 pretreatment groups were 49.76±3.65, 48.21±1.72, 47.99±1.26 respectively, which were significantly lower than that in LPS group (66.88±5.85) (P=0.001, P<0.001, P<0.001). The average fluorescence intensity of p21 in medium and high concentration PLX3397 pretreatment groups were (34.43±3.62, 30.13±0.86), significantly lower than that in LPS group (46.82±5.33) (P=0.043, P=0.007). The expression of p16 protein in low, medium and high concentration PLX3397 pretreatment groups (0.56±0.04, 0.55±0.04, 0.35±0.19) were significantly lower than that in LPS group (0.98±0.10) (P=0.003, P=0.002, P<0.001), as well the expression of p21 protein (0.69±0.20, 0.42±0.08, 0.26±0.14) (P=0.032, P=0.002, P<0.001). According to the results of RT-qPCR, the expressions of IL-6, IL-1β, CXCL-1, CXCL-10 and MMP-8 in PLX3397 pretreatment groups were significantly lower than those in LPS group (P<0.001), while the expression of TGF-β increased (P<0.001). Conclusions: LPS could induce the cell senescence, increase the secretion of SASP and aggravate local inflammation by activating the CSF-1R on the cell surface of bone marrow-derived macrophages. CSF-1R inhibitor PLX3397 might attenuate CSF-1R activation associated with LPS and inhibit the senescence of bone marrow-derived macrophages induced by LPS.
Mice ; Animals ; Male ; Lipopolysaccharides/pharmacology* ; Macrophage Colony-Stimulating Factor/metabolism* ; Matrix Metalloproteinase 8/metabolism* ; Mice, Inbred C57BL ; Macrophages ; Transforming Growth Factor beta/metabolism* ; RNA, Messenger/metabolism*

Objective To explore the effect and mechanism of penehyclidine hydrochloride (PHCD) on vascular endothelial injury in septic rats. Methods Fifty male SD rats were randomly divided into control group, lipopolysaccharide (LPS) induced sepsis group (model group), low dose PHCD (0.3 mg/kg) group, medium dose PHCD (1.0 mg/kg) group and high dose PHCD (3.0 mg/kg) groups, ten mice for each group. Normal saline was injected into the tail vein of the control group, and 10 mg/kg lipopolysaccharide (LPS) was injected into the tail vein of the rats in other groups to prepare the sepsis rat models. After the models were successfully established, low, medium and high doses (0.3, 1.0, 3.0 mg/kg) of PHCD solution were injected into the tail vein of the rats of corresponding groups. Wet/dry mass ratio (W/D) of lung tissue of rats in each group was measured, and ELISA was used to assay interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6 content and rat plasma angiopoietin 2 (Ang2) content in bronchoalveolar lavage fluid (BALF). HE staining was used to observe the pathological changes of lung tissues. Immunohistochemical staining was used to observe the expression of Ang2 in the right lung tissues. Western blot analysis was performed to detect Ang2 and vascular endothelial cadherin (VE-cadherin) protein in lung tissues. Results Compared with the control group, the W/D ratio of the lung tissues of rats in the model group and the contents of IL-1β, IL-6 and TNF-α in BALF were significantly increased; the lung tissues showed obvious pathological damage, with up-regulation of Ang2 expression and down-regulation of VE-Cadherin expression. Compared with the model group, the W/D ratio of the lung tissues of rats in three PHCD treatment groups and the contents of IL-1β, IL-6 and TNF-α in BALF were significantly reduced; the pathological damage of lung tissue was significantly reduced, with down-regulation of Ang2 expression and up-regulation of VE-cadherin expression. Conclusion PHCD can reduce LPS-induced lung inflammation in rats with sepsis by regulating the Ang2/VE-Cadherin pathway, thereby improving vascular endothelial injury.
Rats ; Mice ; Animals ; Male ; Lipopolysaccharides/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Angiopoietin-2/pharmacology* ; Interleukin-6/metabolism* ; Rats, Sprague-Dawley ; Lung ; Acute Lung Injury/metabolism* ; Sepsis/metabolism*

OBJECTIVE: To investigate the role of platelet-rich plasma (PRP) in inducing the M2 macrophage polarization via regulating AMPK singling pathway. METHODS: The expressions of M1 marker CD11c and M2 marker CD206 in macrophages of blank control group, LPS group, LPS+PRP group, and LPS+PRP+Compound C group were detected by flow cytometry. Western blot was used to observe the effects of PRP on the expression of AMPK-mTOR signaling pathway-related proteins at different times (12 h, 18 h and 24 h) after LPS treatment. RNA interference technology was used to silence the expression of AMPK in macrophages, and the expression of TGF-β protein was subsequently examined by Western blot. RESULTS: LPS significantly reduced the expression of CD206 and increased the expression of CD11c (P <0.05). After the addition of PRP, the expression of CD206 was significantly increased (P <0.05), while the expression of CD11c was significantly decreased (P <0.05). Compared with LPS group, PRP treatment significantly increased the expressions of p-AMPK and p-ULK1 proteins at 12 h, 18 h and 24 h, while significantly decreased the expression of p-mTOR protein (P <0.05). After the addition of AMPK inhibitor Compound C, the expression of CD206 was significantly reduced (P <0.05) and the expression of CD11c was significantly increased compared with LPS+PRP group (P <0.05). After silencing the expression of AMPK in macrophages, the promotion effect of PRP on TGF-β was significantly reduced (P <0.05). CONCLUSION PRP can stimulate the transformation of macrophages to M2 type via AMPK signalling pathway.
Humans ; AMP-Activated Protein Kinases/pharmacology* ; Lipopolysaccharides/pharmacology* ; Macrophages/metabolism* ; Transforming Growth Factor beta/metabolism* ; Platelet-Rich Plasma/metabolism*