OBJECTIVETo investigate the effects of TNF-alpha, TNF-beta and the acceptor expression about mechanical renal trauma with extraneous ADM.

METHODSThere were 104 healthy adult plain grade Wistar rat, randomly divided into four groups:8 in the group of control, 32 in the group of trauma, 32 in the group injected ADM before trauma, 32 in the group injected ADM post trauma. The experimental model of rat kidney with mechanical trauma was prepared by striking the area of rat skin reflecting by kidney with free dropping ferrous hammer in the last three groups. ADM (0.1 nmol/kg) administrated by intraperitoneal injection at 10 minutes before trauma or post trauma respectively in injected groups. All rats were executed by drawing-out all the blood in their hearts. Renal tissue was investigated to study positive expression of TNF-alpha, TNF-beta, TNFR after SABC stained.

RESULTSTNF-alpha expression:the TNF-alpha expression of trauma group was more positive than it of control group in the wound early time. The expression of group injected post trauma was less than it of trauma group at 1 h (P < 0.01). The expression of group injected before trauma was less than it of trauma group at 6 h (P < 0.05) TNF-beta expression: the TNF-beta expression of trauma group was less than it of control group at 1 h and 6 h (P < 0.05). The TNF-beta expression of group injected post trauma was more positive than it of trauma group at the same time of 1 h and 6 h (P < 0.01). TNFR expression: the TNFR expression of trauma group was less than it of control group at 6 h (P < 0.01). The TNFR expression of group injected before trauma was more positive than it of trauma group in the at the same time of 1 h and 6 h (P < 0.01).

CONCLUSIONSThe TNFR can regulate the TNF-alpha and the TNF-beta in dynamic balancing. The regulation of TNFR is main to TNF-alpha. What the TNF-beta participated in renal trauma mainly is the anti-damage process. ADM can reduce the expression of TNF-alpha. ADM increases the expression of TNF-beta and TNFR.

Adrenomedullin ; pharmacology ; Animals ; Disease Models, Animal ; Female ; Kidney ; injuries ; metabolism ; Lymphotoxin-alpha ; metabolism ; Male ; Rats ; Rats, Wistar ; Receptors, Tumor Necrosis Factor ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Objective: To investigate the effect of microRNA (miR-148b) targeting decoy receptor 3 (DcR3) on macrophage polarization in sepsis. Methods: Experimental study. From December 2019 to December 2022, serum microRNA expression was detected in 3 patients with sepsis and 3 healthy controls in the clinical laboratory of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Phorbol 12-myristate 13-acetate (PMA) was used to induce the differentiation of human acute monocytic leukemia cells THP-1 into macrophages, and then lipopolysaccharide (LPS) was added to stimulate the establishment of a sepsis cell model, and the expression changes of miR-148b and DcR3 were detected by RT-PCR and Western blot. Overexpression of DcR3 was used to detect the expression levels of TNF-α, CD163 and IL-10 in macrophages stimulated by LPS (100 ng/ml). Overexpression of miR-148b was used to observe the changes of molecular markers of macrophage polarization. The targeting regulation effect of miR-148b on DcR3 was determined by dual-luciferase reporter assay. t test was used to analyze whether there were statistical differences among the groups. Results: The expression of miR-148b was down-regulated (P<0.05) and the expression of DcR3 was up-regulated (P<0.01) in THP-1 macrophages stimulated by LPS. Overexpression of DcR3 inhibited the expression of TNF-α (P<0.05) and promoted the expression of CD163 (P<0.01) and IL-10 (P<0.01). When miR-148b mimics was added, the opposite effect was observed. The dual-luciferase reporter assay confirmed that miR-148b targets and binds to DcR3, inhibiting its transcription and expression. The results of flow cytometry showed that DcR3 could reverse the promoting effect of miR-148b on the CD86/CD163 ratio of macrophages (P<0.05). Conclusion: miR-148b inhibits the expression of DcR3, thereby inhibiting M2 polarization in LPS-stimulated macrophage cells.
Humans ; Interleukin-10 ; Lipopolysaccharides/pharmacology* ; Macrophages ; MicroRNAs/genetics* ; Receptors, Tumor Necrosis Factor, Member 6b/metabolism* ; Tumor Necrosis Factor-alpha

Objective: To investigate the effect of microRNA (miR-148b) targeting decoy receptor 3 (DcR3) on macrophage polarization in sepsis. Methods: Experimental study. From December 2019 to December 2022, serum microRNA expression was detected in 3 patients with sepsis and 3 healthy controls in the clinical laboratory of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Phorbol 12-myristate 13-acetate (PMA) was used to induce the differentiation of human acute monocytic leukemia cells THP-1 into macrophages, and then lipopolysaccharide (LPS) was added to stimulate the establishment of a sepsis cell model, and the expression changes of miR-148b and DcR3 were detected by RT-PCR and Western blot. Overexpression of DcR3 was used to detect the expression levels of TNF-α, CD163 and IL-10 in macrophages stimulated by LPS (100 ng/ml). Overexpression of miR-148b was used to observe the changes of molecular markers of macrophage polarization. The targeting regulation effect of miR-148b on DcR3 was determined by dual-luciferase reporter assay. t test was used to analyze whether there were statistical differences among the groups. Results: The expression of miR-148b was down-regulated (P<0.05) and the expression of DcR3 was up-regulated (P<0.01) in THP-1 macrophages stimulated by LPS. Overexpression of DcR3 inhibited the expression of TNF-α (P<0.05) and promoted the expression of CD163 (P<0.01) and IL-10 (P<0.01). When miR-148b mimics was added, the opposite effect was observed. The dual-luciferase reporter assay confirmed that miR-148b targets and binds to DcR3, inhibiting its transcription and expression. The results of flow cytometry showed that DcR3 could reverse the promoting effect of miR-148b on the CD86/CD163 ratio of macrophages (P<0.05). Conclusion: miR-148b inhibits the expression of DcR3, thereby inhibiting M2 polarization in LPS-stimulated macrophage cells.
Humans ; Interleukin-10 ; Lipopolysaccharides/pharmacology* ; Macrophages ; MicroRNAs/genetics* ; Receptors, Tumor Necrosis Factor, Member 6b/metabolism* ; Tumor Necrosis Factor-alpha

Cell Line ; Glucose ; metabolism ; pharmacology ; Humans ; Mesangial Cells ; drug effects ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Inflammation underlies a wide variety of physiological and pathological processes, and plays a pivotal role in controlling pathogen infection. C1q/tumor necrosis factor (TNF) related proteins (CTRPs), a newly discovered adipokine family with conservative structure and wide distribution, has attracted increasing attention. The CTRP family consists of more than 15 members which fall into the characteristic C1q domain. Increasing studies have demonstrated that CTRPs are involved in the onset and development of inflammation and metabolism as well as related diseases, including myocardial infarction, sepsis and tumors. Here, we first clarified the characteristic domains of CTRPs, and then elucidated their roles in inflammatory-related diseases. Taken together, the information presented here provides new perspectives for therapeutic strategies to improve inflammatory and metabolic abnormalities.
Humans ; Complement C1q/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Inflammation/metabolism* ; Myocardial Infarction

Cell Line ; Humans ; Lipid Metabolism ; Neurons ; metabolism ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE: To investigate the role of acetylated modification induced by coactivator p300 in lipopolysaccharide (LPS)- induced inflammatory mediator synthesis and its molecular mechanism. METHODS: Agilent SurePrint G3 Mouse Gene Expression V2 microarray chip and Western blotting were used to screen the molecules whose expression levels in mouse macrophages (RAW246.7) were correlated with the stimulation intensity of LPS. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (chip-qPCR) were used to verify the binding of the molecules to the promoters of IL-6 and TNF-α genes. The effects of transfection of RAW246.7 cells with overexpression or interfering plasmids on IL-6 and TNF-α synthesis were evaluated with ELISA, and the binding level of the target molecules and acetylation level of H3K27 in the promoter region of IL-6 and TNF-α genes were analyzed by chromatin immunoprecipitation sequencing technique (chip-seq). RESULTS: Gene microarray chip data and Western blotting both confirmed a strong correlation of p300 expression with the stimulation intensity of LPS. Immunocoprecipitation confirmed the binding between p300 and c-myb. The results of EMSA demonstrated that c-myb (P < 0.05), but not p300, could directly bind to the promoter region of IL-6 and TNF-α genes; p300 could bind to the promoters only in the presence of c-myb (P < 0.05). The expressions of p65, p300 and c-myb did not show interactions. Both p300 overexpression and LPS stimulation could increase the level of promoter-binding p300 and H3K27 acetylation level, thus promoting p65 binding and inflammatory gene transcription; such effects were obviously suppressed by interference of c-myb expression (P < 0.05). Interference of p65 resulted in inhibition of p65 binding to the promoters and gene transcription (P < 0.05) without affecting p300 binding or H3K27 acetylation level. CONCLUSION LPS can stimulate the synthesis of p300, whose binding to the promoter region of inflammatory genes via c-myb facilitates the cohesion of p65 by inducing H3K27 acetylation, thus promoting the expression of the inflammatory genes.
Acetylation ; Animals ; Inflammation Mediators ; Interleukin-6/metabolism* ; Lipopolysaccharides/pharmacology* ; Mice ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVE: To investigate the effects of Tongxinluo (TXL) on thromboangiitis obliterans (TAO) and the underlying mechanisms. METHODS: Ninety male C57/BL6J mice were randomly divided into 6 groups according to a random number table: the sham group, TAO model group, Compound Danshen Tablet (CDT) group, and the high-, medium-, and low-dose TXL groups. All mice except the sham group were injected with sodium laurate (0.1 mL, 5 mg/mL) in the femoral artery to establish TAO mouse model. After modeling, mice in the sham and TAO model groups were intragastrically administered 0.5% (w/v) sodium carboxymethylcellulose, mice in the CDT group were intragastrically administered 0.52 g/kg CDT, and mice in the TXL-H, TXL-M, and TXL-L groups were intragastrically administered 1.5, 0.75, and 0.38 g/kg TXL, respectively. After 4 weeks of gavage, the recovery of blood flow in the lower limbs of mice was detected by Laser Doppler Imaging. The pathological changes and thrombosis of the femoral artery were observed by morphological examination. The expressions of tumor necrosis factor α (TNF-α) and inducible nitric oxide synthase (iNOS) in the femoral artery wall were detected by HE staining. Levels of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (6-keto-PGF1α), endothelin-1 (ET-1), interleukin (IL)-1β and IL-6 were measured using enzyme-linked immunosorbent assay (ELISA). Levels of activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) were detected by a fully automated biochemical analyzer. RESULTS: TXL promoted the restoration of blood flow in the lower limbs, reduced the area of thrombosis in the femoral artery, and alleviated the pathological changes in the femoral artery wall. Moreover, the levels of TXB2, ET-1, IL-6, IL-1β, TNF-α and iNOS were significantly lower in the TXL groups compared with the model group (P<0.05 or P<0.01), while the level of 6-keto-PGF1α was significantly higher (P<0.01). In addition, APTT, PT, and TT were significantly prolonged in TXL groups compared with the model group (P<0.05 or P<0.01), and FIB levels were significantly decreased compared with the model group (P<0.01). CONCLUSIONS TXL had a protective effect on TAO mice, and the mechanism may involve inhibition of thrombosis and inflammatory responses. TXL may be a potential drug for the treatment of TAO.
Mice ; Male ; Animals ; Thromboangiitis Obliterans/chemically induced* ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Thrombosis

OBJECTIVE: To explore the role of the low-affinity A2b adenosine receptors (Adora2b) in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide and its mechanism. METHODS: Rat pulmonary microvascular endothelial cells (PMVECs) were isolated and cultured in vitro. After serum deprivation for 24 hours, cells were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then challenged with LPS (100 μg/L). Cells without treatment were served as the control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. After incubation with specific drugs for 24 hours, the apoptosis of PMVECs was analyzed by flow cytometry using Annexin V/propidium iodide (PI) technique. The levels of early inflammatory factors in cultured medium were measured using enzyme linked immunosorbent assay (ELISA). The mRNA expressions of chemotactic factors and adhesion molecules were determined by real-time quantitative-polymerase chain reaction (RT-qPCR). Polymorph nuclear neutrophils (PMNs) from venous blood of healthy rats were isolated, and PMN migration through PMVECs monolayer under stimulation of drugs was observed in transwell inserts. The monolayer permeability of PMVECs after adhesion of PMNs was determined by fluorescein isothiocyanate (FITC)-albumin assay. Oxidative stress was detected by DCFH-DA assay. RESULTS: Compared with the control group, more cells entered into the apoptosis stage after LPS challenge. Meanwhile, the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cultured medium were significantly increased, as well as the mRNA expressions of chemotactic factors [C-X-C motif chemokine ligand 1 (CXCL-1), CXCL-3 and monocyte chemoattractant protein-1 (MCP-1)] and adhesion molecules [E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. More PMNs migrated through PMVECs following adhesion and the monolayer permeability of PMVECs was rapidly enhanced. The oxidative stress was upregulated. Compared with LPS group, BAY60-6583 pretreatment could dose-dependently decrease the rate of apoptosis, attenuate trans-endothelial migration of PMNs and decrease the endothelial cell barrier leakage. There were significant differences even after incubation of 0.1 μmol/L BAY60-6583 [apoptosis rate: (21.12±2.12)% vs. (27.66±3.57)%, number of migrated PMNs/HP: 260.60±18.24 vs. 290.20±16.48, permeability coefficient (Pd, ×10^-6 cm/s): 28.28±2.04 vs. 32.55±2.13, all P < 0.05]. Meanwhile, BAY60-6583 pretreatment also downregulated the levels of early proinflammatory factors in a dose-dependent manner as well as the mRNA expressions of chemotactic factors and adhesion molecules. The statistic difference was significant while treated with 1 μmol/L BAY60-6583 [IL-1β (ng/L): 475.75±63.15 vs. 755.25±67.42, TNF-α (ng/L): 560.25±69.96 vs. 818.75±60.92, CXCL-1 mRNA (2^-ΔΔCt): 3.57±0.28 vs. 5.27±0.69, CXCL-3 mRNA (2^-ΔΔCt): 4.56±0.48 vs. 7.32±0.54, MCP-1 mRNA (2^-ΔΔCt): 2.21±0.31 vs. 3.35±0.21, E-selectin mRNA (2^-ΔΔCt): 4.64±0.09 vs. 7.28±0.73, ICAM-1 mRNA (2^-ΔΔCt): 4.14±0.30 vs. 5.89±0.25, VCAM-1 mRNA (2^-ΔΔCt): 2.23±0.19 vs. 2.92±0.33, all P < 0.05]. Furthermore, pretreatment of 10 μmol/L BAY60-6583 could decrease the oxidative stress [reactive oxygen species (RFU): 629.05±33.10 vs. 781.45±64.59, P < 0.05]. Contrast, PSB1115 pretreatment aggravated apoptosis of PMVECs after LPS incubation [(34.36±4.57)% vs. (27.66±3.57)%], upregulated expressions of proinflammatory and chemotactic factors as well as adhesion molecules [IL-1β (ng/L): 889.00±63.11 vs. 755.25±67.42, TNF-α (ng/L): 939.00±43.44 vs. 818.75±60.92, CXCL-1 mRNA (2^-ΔΔCt): 6.66±0.65 vs. 5.27±0.69, CXCL-3 mRNA (2^-ΔΔCt): 10.42±0.51 vs. 7.32±0.54, MCP-1 mRNA (2^-ΔΔCt): 4.85±0.34 vs. 3.35±0.21, E-selectin mRNA (2^-ΔΔCt): 8.42±0.47 vs. 7.28±0.73, ICAM-1 mRNA (2^-ΔΔCt): 7.46±0.72 vs. 5.89±0.25, VCAM-1 mRNA (2^-ΔΔCt): 4.35±0.26 vs. 2.92±0.33], aggravated trans-endothelial migration of PMNs (cells/HP: 348.40±22.68 vs. 290.20±16.48), enhanced the leakage of PMVECs monolayer [Pd (×10^-6 cm/s): 39.65±2.69 vs. 32.55±2.13] and increased oxidative stress in PMVECs [reactive oxygen species (RFU): 847.04±29.26 vs. 781.45±64.59], with statistically significant difference (all P < 0.05). CONCLUSIONS Activation of endothelial Adora2b attenuates LPS-induced pulmonary microvascular inflammation by decreasing the release of early inflammatory factors, downregulating expressions of chemotactic factors and adhesion molecules, attenuating trans-endothelial migration of PMNs and oxidative stress in PMVECs, which suggest endothelial Adora2b is apotential anti-inflammatory target in the treatment of LPS-induced acute lung injury.
Animals ; Endothelial Cells ; Inflammation ; Lipopolysaccharides/metabolism* ; Pneumonia ; Rats ; Receptor, Adenosine A2B/metabolism* ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the expression of nuclear factor kappa B (NF-kgr;B) and tumor necrosis factor &alpha; (TNF-&alpha;) in lung tissue of acute paraquat poisoned rats.

METHODS68 male Wistar rats were randomly divided into 2 groups: the control group (n = 8), the intoxication group (n = 60). On the 1st, the 3rd, the 7th, the 14th and the 28th day after intoxication, the expression of NF-κB p65 and TNF-&alpha; in lung tissue were detected by LSAB immunohistochemistry (IH) staining. Meanwhile, the level of malondialdehyde (MDA) in plasma, and lung homogenate, the content of malondialdehyde (HPY) in lung homogenate were detected.

RESULTSThe levels of MDA in plasma on the 1st, the 3rd, the 7th day and in lung homogenate on the 1st, the 3rd day of the intoxication group [in plasma: (10.15 ± 3.15), (6.97 ± 1.65) and (5.44 ± 0.66) nmol/ml; in lung homogenate: (10.20 ± 2.43), (10.71 ± 171) nmol/ml] were significantly higher than that of the control group [in plasma: (3.84 ± 1.04) nmol/ml, in lung homogenate: (7.66 ± 0.66) nmol/ml]. The content of HPY in lung homogenate on the 14th and the 28th day after intoxication [(19.98 ± 2.86), (26.06 ± 4.06) µg/0.1 g lung homogenate] were higher than that of the control group [(8.80 ± 1.26) µg/0.1 g lung homogenate] significantly. The expression of NF-κB p65 and TNF-&alpha; in lung tissue were both significantly increased on the first day and the 3rd day of the intoxication group compared with the control group and weakened obviously after the 7th day.

CONCLUSIONAcute paraquat poisoning can induce increased expression of both NF-κB p65 and TNF-&alpha; in lung tissue; the enhanced activity of NF-κB may take part in the process of pulmonary injury in PQ poisoning.

Animals ; Lung ; metabolism ; pathology ; Male ; Paraquat ; poisoning ; Rats ; Rats, Wistar ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism