In order to investigate whether the effect of Yuxuebi Tablets on the peripheral and central inflammation resolution of mice with inflammatory pain is related to their regulation of G protein-coupled receptor 37(GPR37), an inflammatory pain model was established by injecting complete Freund's adjuvant(CFA) into the paws of mice, with a sham-operated group receiving a similar volume of normal saline. The mice were assigned randomly to the sham-operated group, model group, ibuprofen group(91 mg·kg~(-1)), and low-, medium-, and high-dose groups of Yuxuebi Tablets(60, 120, and 240 mg·kg~(-1)). The drug was administered orally from days 1 to 19 after modeling. Von Frey method and the hot plate test were used to detect mechanical pain thresholds and heat hyperalgesia. The levels of interleukin-10(IL-10) and transforming growth factor-beta(TGF-β) in the spinal cord were quantified using enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein expression of GPR37 in the spinal cord was measured by real-time quantitative reverse transcription PCR(qRT-PCR) and Western blot. Additionally, immunofluorescence was used to detect the expression of macrosialin antigen(CD68), mannose receptor(MRC1 or CD206), and GPR37 in dorsal root ganglia, as well as the expression of calcium-binding adapter molecule 1(IBA1), CD206, and GPR37 in the dorsal horn of the spinal cord. The results showed that compared with those of the sham-operated group, the mechanical pain thresholds and hot withdrawal latency of the model group significantly declined, and the expression of CD68 in the dorsal root ganglia and the expression of IBA1 in the dorsal horn of the spinal cord significantly increased. The expression of CD206 and GPR37 significantly decreased in the dorsal root ganglion and dorsal horn of the spinal cord, and IL-10 and TGF-β levels in the spinal cord were significantly decreased. Compared with those of the model group, the mechanical pain thresholds and hot withdrawal latency of the high-dose group of Yuxuebi Tablets significantly increased, and the expression of CD68 in the dorsal root ganglion and IBA1 in the dorsal horn of the spinal cord significantly decreased. The expression of CD206 and GPR37 in the dorsal root ganglion and dorsal horn of the spinal cord significantly increased, as well as IL-10 and TGF-β levels in the spinal cord. These findings indicated that Yuxuebi Tablets may reduce macrophage(microglial) infiltration and foster M2 macrophage polarization by enhancing GPR37 expression in the dorsal root ganglia and dorsal horn of the spinal cord of CFA-induced mice, so as to improve IL-10 and TGF-β levels, promote resolution of both peripheral and central inflammation, and play analgesic effects.
Inflammation/genetics* ; Pain/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Animals ; Mice ; Freund's Adjuvant/pharmacology* ; Ibuprofen ; Pain Threshold/drug effects* ; Hyperalgesia/genetics* ; Ganglia, Spinal ; Interleukin-10/genetics* ; Transforming Growth Factor beta/genetics* ; Reverse Transcriptase Polymerase Chain Reaction ; Tablets ; Receptors, G-Protein-Coupled

Objective To investigate the mechanism by which miR-148a affects M2 macrophage polarization and inhibits liver cancer cell proliferation through Wnt3a/β-catenin. Methods The mRNA expression levels of miR-148a, CD206 and interleukin-10 (IL-10) in tumor tissues and adjacent non-tumor liver tissues of 84 patients with liver cancer were detected by real-time quantitative PCR. THP-1 cells were separated into blank group (conventional culture), M2 group (200 nmol/L phorbol ester, 20 ng/mL IL-4, 20 ng/mL IL-13), M2 combined with negative control (miR-NC) group (transfected with miR-NC on the basis of M2 group), M2 combined with miR-148a mimics (transfected with miR-148a mimics on the basis of M2 group) group, M2 combined with miR-148a mimics combined with Wnt3a (treated with 100 μg/L Wnt3a on top of M2 combined with miR-148a mimics group) group. The proliferation of HuH7 cells was detected by CCK-8 and EdU methods. Apoptosis and M2 macrophage marker CD206 was detected by flow cytometry. The level of IL-10 in cell supernatant was detected by chemiluminescence method; The mRNA levels of miR-148a, CD206 and IL-10 were detected by real-time quantitative PCR. The protein levels of Wnt3a and β-catenin were detected by Western blot. Results The expressions of CD206, IL-10 mRNA, Wnt3a and β-catenin in tumor tissue were higher than those in non-tumor liver tissues, and the miR-148a level was decreased. The mRNA expression of M2 macrophage markers CD206 and IL-10 were significantly increased. Compared with the blank group, the OD450 value, EdU positive rate, the mRNA expressions of CD206 and IL-10, the level of IL-10 in the supernatant, and the expressions of Wnt3a and β-catenin were increased in M2 group, while the apoptotic rate and miR-148a level were decreased. Compared with M2 group and M2 combined with miR-NC group, the OD₄₅₀ value, EdU positive rate, the mRNA expressions of CD206 and IL-10, the level of IL-10 in the supernatant, and the expressions of Wnt3a and β-catenin were decreased in M2 combined with miR-148a mimics group, while the apoptotic rate and miR-148a level were increased. Wnt3a reversed the inhibitory effect of miR-148a overexpression on the proliferation of liver cancer cells. Conclusion Overexpression of miR-148a inhibits M2 polarization of macrophages and prevents the proliferation of liver cancer cells, which may be related to the inhibition of the Wnt3a/β-catenin pathway.
Humans ; MicroRNAs/metabolism* ; Wnt3A Protein/metabolism* ; Liver Neoplasms/metabolism* ; Cell Proliferation/genetics* ; beta Catenin/genetics* ; Macrophages/metabolism* ; Interleukin-10/metabolism* ; Apoptosis/genetics* ; Cell Line, Tumor ; Female ; Male ; Mannose Receptor ; Lectins, C-Type/metabolism* ; Mannose-Binding Lectins/metabolism* ; Middle Aged ; Receptors, Cell Surface/metabolism*

Objective To investigate the effect of ATP synthase inhibitory factor 1 (ATPIF1) on the antitumor activity of chimeric antigen receptor (CAR)-NK92 cells. Methods HER2-targeted CAR-NK92 cells with ATPIF1 overexpression or knockdown were constructed. CAR-positive expression rate was detected by flow cytometry. Cell proliferation capacity was measured using CCK-8 assay. Glycolytic capacity was analyzed by Seahorse metabolic analyzer. Mitochondrial membrane potential levels were detected using JC-1 probe. Target cell lysis rate was evaluated by firefly luciferase reporter assay. Expression levels of CD107a, natural-killer group 2 member D (NKG2D), granzyme B (GzmB), perforin, and interleukin 2 (IL-2) were detected via flow cytometry. Quantitative real-time PCR was used to measure the expression of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), tumor necrosis factor α (TNF-α), ATPIF1, and hexokinase 1 (HK1). The impact of glycolytic inhibition by 2-Deoxy-D-glucose (2-DG) on CAR-NK92 antitumor capacity was examined. Results Successfully generated HER2-targeting control CAR-NK92 cells, as well as ATPIF1-overexpressing and ATPIF1 knockdown CAR-NK92 cells. The ATPIF1-overexpressing CAR-NK92 cells showed significantly enhanced target cell lysis rate, elevated expression levels of NKG2D and CD107a, increased secretion capacities of Granzyme B (GzmB) and IL-2, and upregulated mRNA expression levels of IFIT1 and TNF-α, while ATPIF1-knockdown cells exhibited opposite effects. ATPIF1 overexpression induced metabolic reprogramming in CAR-NK92 cells, manifested by significantly decreased mitochondrial membrane potential (δpsim), markedly upregulated HK1 mRNA expression, and enhanced basal glycolysis and glycolytic capacity. After glycolysis inhibition with 2-DG (5 μmol/L), both ATPIF1-overexpressing and knockdown CAR-NK92 cells showed no significant differences in NKG2D and CD107a expression levels compared to control cells. Conclusion ATPIF1 regulates the antitumor activity of CAR-NK92 cells through modulating glycolytic metabolism. Overexpression of ATPIF1 can enhance the antitumor efficacy of CAR-NK92 cells.
Humans ; Glycolysis ; Killer Cells, Natural/metabolism* ; Receptors, Chimeric Antigen/immunology* ; Granzymes/genetics* ; Hexokinase/metabolism* ; Cell Line, Tumor ; Interleukin-2/genetics* ; Cell Proliferation ; NK Cell Lectin-Like Receptor Subfamily K/genetics* ; Membrane Potential, Mitochondrial

OBJECTIVE: To assess the association of single nucleotide polymorphisms (SNPs) of interleukin-23 receptor (IL-23R) gene with susceptibility to psoriasis. METHODS: Two hundred and ten psoriasis patients admitted to Xinxiang Central Hospital from January 2019 to December 2024 were selected as the study group, and 210 healthy individuals undergoing physical examination during the same period were selected as the control group. 3 mL of peripheral venous blood sample was collected from each individual from the two groups, and PCR-Restriction fragment length polymorphism (PCR-RFLP) assay was used to determine the polymorphisms of the IL-23R gene at rs2201841, rs1004819, rs10889677, rs1343151 and rs1495965 loci. Genotypic and allelic distribution of each SNP locus was calculated to assess the association between SNPs of the IL-23R gene with the onset of psoriasis, and the difference in serum IL-23 levels among patients with different genotypes at each locus was compared. This study was approved by the Medical Ethics Committee of Xinxiang Central Hospital (Ethic No. 2024-749). RESULTS: The results showed that the frequency of CC genotype at rs1004819 locus of the study group was significantly higher than that of the control group (26.19% vs. 18.10%, P < 0.05), and the frequency of C allele was also significantly higher than that of the control group (54.05% vs. 42.62%, P < 0.05). There was no significant difference in allelic and genotypic frequencies between the two groups at rs2201841, rs10889677, rs1343151, and rs1495965 loci (P > 0.05). The dominant and recessive inheritance patterns at the rs1004819 locus are associated with susceptibility to psoriasis (P < 0.05), while the different inheritance patterns at rs2201841, rs10889677, rs1343151, and rs1495965 loci are not associated with psoriasis (P > 0.05). The serum IL-23 levels of patients with CC genotype at the rs1004819 locus were higher than those with the CT and TT genotypes (P < 0.05). No significant difference was detected in the serum levels of IL-23 between patients with different genotypes for the rs2201841, rs10889677, rs1343151, and rs1495965 loci (P > 0.05). CONCLUSION The polymorphism at the rs1004819 locus of the IL-23R gene is associated with susceptibility to psoriasis, and individuals carrying the CC genotype and C allele have a higher risk of developing the disease.
Humans ; Psoriasis/genetics* ; Polymorphism, Single Nucleotide ; Receptors, Interleukin/genetics* ; Genetic Predisposition to Disease ; Male ; Female ; Adult ; Middle Aged ; Genotype ; Alleles ; Gene Frequency ; Case-Control Studies ; Interleukin-23/blood*

Cancer pain is one of the most common symptoms in patients with advanced cancer. In this study, we aimed to investigate the effects of the Mas-related gene C (MrgC) receptors on bone cancer pain. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured after the inoculation of Walker 256 mammary gland carcinoma cells into the tibia of adult Sprague-Dawley rats. The effects of MrgC receptor agonist bovine adrenal medulla 8-22 (BAM8-22) on nociceptive behaviors were investigated after intrathecal injection on days 16 and 17. Glial fibrillary acidic protein (GFAP)-positive cells in the spinal dorsal cord, and calcitonin gene related peptide (CGRP)-, neuronal nitric oxide synthase (nNOS)- and IL-1β-positive neurons in the dorsal root ganglia (DRG) were examined by immunofluorescence staining. The expression of nNOS and IL-1β proteins in the spinal dorsal horn and the DRG was examined by Western blotting after treatment with (Tyr6)-γ2-MSH-6-12 (MSH), which was another MrgC receptor agonist. The results showed that intrathecal injection of BAM8-22 (30 nmol) attenuated mechanical allodynia in a rat model of bone cancer pain and the effects could last for about 60 min, and single administration of BAM8-22 for two consecutive days reduced mechanical allodynia by about half on the third day. Moreover, the number of GFAP-positive cells in the spinal dorsal horn, and the number of CGRP-, nNOS- and IL-1β-positive neurons in the DRG were decreased. Similarly, intrathecal administration of MSH (15 nmol) reduced the expression of nNOS and IL-1β in the spinal dorsal horn and the DRG. In conclusion, activation of MrgC receptors suppresses the activation of astrocytes in the spinal dorsal cord and the expression of CGRP, nNOS, and IL-1β in the spinal dorsal cord and/or DRG, which may underlie the inhibition of bone cancer pain. These findings provide a novel strategy for the treatment of bone cancer pain.
Animals ; Cancer Pain/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Bone Neoplasms/complications* ; Ganglia, Spinal/metabolism* ; Spinal Cord Dorsal Horn/metabolism* ; Receptors, G-Protein-Coupled/genetics* ; Female ; Calcitonin Gene-Related Peptide/genetics* ; Interleukin-1beta/metabolism* ; Peptide Fragments/metabolism* ; Nitric Oxide Synthase Type I/genetics* ; Disease Models, Animal

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 This study aims to construct and identify the chimeric antigen receptor NK92 (CAR-NK92) cells targeting NKG2D ligand (NKG2DL) (secreting IL-15Ra-IL-15) and verify the killing activity of NKG2D CAR-NK92 cells against multiple myeloma cells. Methods The extracellular segment of NKG2D was employed to connect 4-1BB and CD3Z, as well as IL-15Ra-IL-15 sequence to obtain a CAR expression framework. The lentivirus was packaged and transduced into NK92 cells to obtain NKG2D CAR-NK92 cells. The proliferation of NKG2D CAR-NK92 cells was detected by CCK-8 assay, IL-15Ra secretion was detected by ELISA and killing efficiency was detected by lactate dehydrogenase (LDH) assay. The molecular markers of NKp30, NKp44, NKp46, the ratio of apoptotic cell population, CD107a, and the secretion level of granzyme B and perforin were detected using flow cytometry. In addition, the cytotoxic mechanism of NKG2D CAR-NK92 cells on the tumor was verified by measuring the degranulation ability. Moreover, after NKG2D antibody inhibited effector cells and histamine inhibited tumor cells, LDH assay was utilized to detect the effect on cell-killing efficiency. Finally, the multiple myeloma tumor xenograft model was constructed to verify its anti-tumor activity in vivo. Results Lentiviral transduction significantly increased NKG2D expression in NK92 cells. Compared with NK92 cells, the proliferation ability of NKG2D CAR-NK92 cells was weaker. The early apoptotic cell population of NKG2D CAR-NK92 cells was less, and NKG2D CAR-NK92 cells had stronger cytotoxicity to multiple myeloma cells. Additionally, IL-15Ra secretion could be detected in its culture supernatant. NKp44 protein expression in NKG2D CAR-NK92 cells was clearly increased, demonstrating an enhanced activation level. Inhibition test revealed that the cytotoxicity of CAR-NK92 cells to MHC-I chain-related protein A (MICA) and MICB-positive tumor cells was more dependent on the interaction between NKG2D CAR and NKG2DL. After stimulating NKG2D CAR-NK92 cells with tumor cells, granzyme B and perforin expression increased, and NK cells obviously upregulated CD107α. Furthermore, multiple myeloma tumor xenograft model revealed that the tumors of mice treated with NKG2D CAR-NK92 cells were significantly reduced, and the cell therapy did not sensibly affect the weight of the mice. Conclusion A type of CAR-NK92 cell targeting NKG2DL (secreting IL-15Ra-IL-15) is successfully constructed, indicating the effective killing of multiple myeloid cells.
Humans ; Mice ; Animals ; Receptors, Chimeric Antigen/genetics* ; Interleukin-15 ; NK Cell Lectin-Like Receptor Subfamily K/metabolism* ; Granzymes ; Cell Line, Tumor ; Multiple Myeloma/therapy* ; Perforin

OBJECTIVE: To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML. METHODS: Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR). RESULTS: Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05). CONCLUSION Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.
Humans ; U937 Cells ; Cytarabine/therapeutic use* ; Receptors, Interleukin-8A ; NF-kappa B ; Proto-Oncogene Proteins c-akt ; Phosphatidylinositol 3-Kinases ; Leukemia, Myeloid, Acute/genetics* ; Apoptosis ; Cell Proliferation ; Apoptosis Regulatory Proteins ; Proto-Oncogene Proteins c-bcl-2 ; RNA, Messenger ; Cell Line, Tumor

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.
Humans ; Female ; Receptors, Chimeric Antigen/genetics* ; Carcinoma, Non-Small-Cell Lung ; CD8-Positive T-Lymphocytes ; Interleukin-2/pharmacology* ; Tumor Necrosis Factor-alpha ; Cell Line, Tumor ; HEK293 Cells ; Lung Neoplasms ; Ovarian Neoplasms ; Immunotherapy, Adoptive

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