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1.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 176-186, 2024.
Artigo em Chinês | WPRIM | ID: wpr-1016477

RESUMO

ObjectiveThrough the correlation analysis between intestinal absorption profile and inhibition of macrophage foaming, the pharmacodynamic components of Zhuriheng dripping pills(ZRH) were explored to provide a basis for establishing its quality standard. MethodIntestinal absorption fluids with 0, 5, 10, 15, 20 times clinical equivalent doses were prepared by a rat everted gut sac(EGS), and the oxidized low density lipoprotein(ox-LDL)-induced RAW264.7 macrophage foaming model was used to investigate the effect of intestinal absorption fluid with different doses on the accumulation of lipids in RAW264.7 cells by oil red O staining and cholesterol content determination, and to screen for the optimal dose. Ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry(UPLC-Q-Exactive Orbitrap MS) was used to analyze and identify intestinal absorption fractions of ZRH intestinal absorption fluids, and partial least squares-discriminant analysis(PLS-DA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were performed on different doses of ZRH intestinal absorption fluids using SIMCA 13.0 with peak area as the independent variable and the pharmacodynamic indicators as the dependent variables to screen the compounds with variable importance in the projection(VIP) value>1.0 as contributing components, and Pearson correlation analysis was used to determine the spectral effect relationship, determined the compounds and positive correlation with pharmacodynamic were as active ingredients. Molecular docking was used to verify the binding energy of peroxisome proliferator-activated receptor α(PPARα), PPARγ, PPARβ, human retinoid X receptor α(RXRA) and nuclear transcription factor-κB(NF-κB) with the active ingredients in ZRH intestinal absorption fluids. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) was performed to detect the mRNA levels of PPARγ, scavenger receptor A1(SRA1) and adenosine triphosphate-binding cassette transporter A1(ABCA1) in RAW264.7 cells, Westen blot was used to detect the expression level of PPARγ protein in RAW264.7 cells, and enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of interleukin(IL)-1β and NF-κB in RAW264.7 cells. ResultAccording to the results of oil red O staining and cholesterol content determination, the ZRH intestinal absorption fluids could significantly reduce macrophage foaming, and intestinal absorption fluids with 15, 20 times clinical equivalent doses had the best effect, the 15-fold ZRH intestinal absorption fluid was finally determined as the study subject. Spectral effect relationship showed that 52 corresponding peaks in the ZRH-containing intestinal fluid were positively correlated with the efficacy, including organic acids, phenylpropanoids, iridoids, flavonoids, bile acids, coumarins and chromones. Target validation results showed that 86.9%-96.2% of the total components processed good binding activities with the key targets of PPARα, PPARγ, PPARβ, RXRA and NF-κB, and the docking energy values were all less than -6.0 kcal·mol-1(1 cal≈4.19 J). The results of validation showed that, compared with the normal group, the model group showed a significant increase in the levels of SRA1 and PPARγ mRNA expression, a significant decrease in ABCA1 mRNA expression, a significant increase in the level of PPARγ protein expression, and a significant increase in the levels of IL-1β and NF-κB(P<0.01), compared with the model group, the 15-fold intestinal absorption fluid group showed a significant decrease in the levels of SRA1 and PPARγ mRNA expression(P<0.05, P<0.01), ABCA1 mRNA expression level was significantly up-regulated, the levels of IL-1β and NF-κB were significantly reduced(P<0.01), and PPARγ protein expression level was significantly reduced(P<0.05). ConclusionThis study identifies 52 components and their metabolites in ZRH intestinal absorption fluid that are positively correlated with the inhibition of macrophage foaming, which may be related to the regulation of the PPARs pathway in cells and the reduction of the levels of inflammatory factors, and can provide a reference for the quality control and clinical application of ZRH.

2.
Chinese Journal of Endocrinology and Metabolism ; (12): 499-505, 2023.
Artigo em Chinês | WPRIM | ID: wpr-994352

RESUMO

Objective:To investigate the effects of myeloid-derived growth factor(MYDGF) on inflammatory response and osteoclast differentiation of RAW264.7 cells.Methods:The RAW264.7 osteoclast precursor cells were cultured and treated with different concentrations of recombinant MYDGF protein(rMYDGF), and their cell viability was assessed using the MTT assay. RAW264.7 cells were induced with lipopolysaccharide(LPS) to induce inflammation, and the expression of inflammatory mediators and cell polarization were observed after intervention with rMYDGF. The RAW264.7 cells were induced for osteoclast differentiation using receptor activator of nuclear factor-κB ligand(RANKL), and rMYDGF was added for intervention. Osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase(TRAP) staining. The osteoclast resorption pits and the number of actin rings(F-actin rings) were observed under a microscope. Reverse transcription PCR was performed to detect the expression of activated T cell nuclear factor 1(Nfatc1), cathepsin K(CTSK), and c-Fos genes during osteoclast differentiation. The protein phosphorylation levels of nuclear factor-κB(NF-κB) signaling pathway proteins were detected using Western blotting.Results:MTT assay showed that rMYDGF did not significantly inhibit the viability of RAW264.7 cell when the concentration was lower than 100 ng/mL. Moreover, rMYDGF inhibited the expression levels of inflammatory factors and M1 cell polarization after LPS stimulation. Compared with the control group, the number and area of TRAP positive cells, the number and area of bone resorption pit were decreased in rMYDGF intervention group respectively, as well as the area of the F-actin ring was reduced and its shape was incomplete after rMYDGF intervention. Furthermore, rMYDGF reduced the expression levels of osteoclast-specific marker genes and inhibited the phosphorylation of NF-κB signaling pathway protein IκBα during osteoclast differentiation.Conclusion:MYDGF inhibits the inflammatory response and osteoclast differentiation of RAW264.7 cells.

3.
Chinese Journal of Biochemistry and Molecular Biology ; (12): 880-888, 2023.
Artigo em Chinês | WPRIM | ID: wpr-1015623

RESUMO

Tumor cells can use different strategies to suppress the immune system and disable them for killing tumor cells. Previous studies have shown that recombinant human peroxiredoxin-5 (hPRDX5) can activate the normal anti-tumor immune, so as to control and eliminate the tumor cells, but its exact mechanism of action needs to be studied in depth. The study aimed to investigate whether hPRDX5 exerts its anti-tumor activity by activating or reversing the polarization state of mouse macrophages RAW264. 7 cells. The results of CCK8 showed that different doses of hPRDX5 could significantly enhance the viability of macrophage compared with the control group (P < 0. 001); The results of Nitric oxide (NO) test showed that hPRDX5 significantly enhanced NO secretion levels in RAW264. 7 cells (P < 0. 001); ELISA experiments revealed that hPRDX5 promotes TNF-α (P<0. 01) and IL-6 (P<0. 001) secretion in RAW264. 7 cells; Flow cytometry revealed that hPRDX5 increased the expression of antigen differentiation cluster (CD) 80 (P < 0. 01) and inducible nitric oxide oxide synthase (iNOS) (P < 0. 001) in RAW264. 7 cells, and reduced the expression of CD206 (P < 0. 001) in RAW264. 7 cells induced by tumor conditional culture solution (TCS); Lactate dehydrogenase (LDH) experiments revealed that hPRDX5 can increase the killing activity of mouse macrophages on mouse pancreatic cancer Panc02 cells. hPRDX5 is able to activate mouse macrophage RAW264. 7 cells, promotes its M1-type polarization, reverses M2-type polarization, and exerts antitumor activity through the immune-enhancing effect.

4.
Chinese Pharmacological Bulletin ; (12): 1296-1302, 2023.
Artigo em Chinês | WPRIM | ID: wpr-1013930

RESUMO

Aim To investigate the effect of the aryl hydrocarbon receptor (AhR) on the expression of inflammatory factors in macrophages RAW264. 7 induced by pyocyanin (PCN) and the regulatory mechanism of its signaling pathway. Methods RAW264. 7 cells were treated with different concentrations of PCN for 24 h, respectively, and the effect of PCN on cell activity was detected by CCK8 assay to determine the optimal PCN concentration for manufacturing infection models. The cells were divided into the control group (given 0. 1% dimethyl sulfoxide DMSO), PCN group, PCN + AhR inhibitor (CH223191) group, and PCN + AhR agonist (FICZ) group, and the expression of AhR was detected by immunofluorescence. The expression levels of inflammatory factors (IL-6, IL-1β, and TNF-α) were detected by ELISA. The protein expression of AhR, pp38 MAPK and p-p65NF-κB, was detected by Western blotting. Results PCN induced a significant quantitative effect on AhR expression in RAW264. 7 cells. CH223191 increased PCN-induced inflammatory factor secretion and enhanced the phosphorylation of p38MAPK and p65NF-κB compared with the control group. FICZ decreased PCN-induced inflammatory factor production and reduced the phosphorylation of p38MAPK and p65NF-κB phosphorylation capacity. Conclusions AhR can regulate PCN-induced inflammatory factor expression in RAW264. 7 cells, and the p38MAPK/p65NF-κB signaling pathway may be an essential pathway for the involvement of AhR in immune regulation.

5.
Chinese Pharmacological Bulletin ; (12): 130-138, 2023.
Artigo em Chinês | WPRIM | ID: wpr-1013889

RESUMO

Aim To investigate the anti-inflammatory effect of L-Shikonin ( SK ) on lipopolysaccharide ( LPS)-induced RAW 264. 7 macrophages in vitro and its protective effect on LPS/D-GalN-induced acute liver injury. Methods The mouse model of acute liver in¬jury was established in vivo experiments by LPS/D- GalN. The survival rate of the mice and the changes of liver and spleen indices in each group were examined. The levels of AST, ALT and AKP in serum and NO, superoxide dismutase ( SOD ) and malondialdehyde (MDA) in liver tissue homogenate were measured, and the histopathological sections of the liver of each group were observed by H&E staining. M I T colorimet- ric assay was used for cell viability in vitro experi¬ments, Griess method for the detection of NO content, RT-PCR assay and Western blot assay for examining the effect of levulinic acid on the expression levels of mRNA and related pathway proteins of pro-inflammato¬ry factors in LPS-induced RAW264. 7 cells. Results The results of in vivo experiments showed that L-SK significantly improved the liver and spleen indices, de¬creased AST, ALT and AKP levels in serum, de¬creased NO and MDA in liver homogenate, and in¬creased SOD activity in mice with acute liver injury. The results of in vitro experiments showed that L-SK significantly inhibited the mRNA expression of INOS, COX2, I FN-(3 and pro-inflammatory factors 1L-6, TNF-a and IL-10 in LPS-induced RAW264. 7 cells, and significantly inhibited the protein expression of IN¬OS, COX2 and the NF-kB signaling pathway. Conclu¬sions L-SK has good anti-inflammatory effects in LPS-induced inflammation in RAW 264. 7 cells in vitro. Il inhibits the protein expression of phosphorylated P65 and IKKaαβ in the NF-kB signaling pathway, thereby suppressing the anti-inflammatory effects in vitro and L- Shikonin has protective effects against acute liver injury in mice.

6.
Chinese Pharmacological Bulletin ; (12): 1711-1717, 2023.
Artigo em Chinês | WPRIM | ID: wpr-1013708

RESUMO

Aim To examine the effect of peptide P3 on lipid accumulation in RAW264.7 cells and the underlying molecular mechanism. Methods MTT method was used to screen the concentration of peptide P3 and oxidized low density lipoprotein(ox-LDL),and RAW.264.7 cells were induced to form foam cells by ox-LDL with 80 mg·L

7.
Chinese Journal of Natural Medicines (English Ed.) ; (6): 852-858, 2023.
Artigo em Inglês | WPRIM | ID: wpr-1010996

RESUMO

We reported the discovery of six novel coumarins, toddasirins A-F (1-6), each endowed with modified isoprenyl or geranyl side chains, derived from the roots of Toddalia asiatica. Comprehensive structural elucidation was achieved through multispectroscopic analyses, single-crystal X-ray diffraction experiments, and advanced quantum mechanical electronic circular dichroism (ECD) calculations. Furthermore, the anti-inflammatory activity of these compounds was assessed. Notably, compounds 1-3 and 6 demonstrated notable inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells, with 50% inhibitory concentration (IC50) values of 3.22, 4.78, 8.90, and 4.31 μmol·L-1, respectively.


Assuntos
Camundongos , Animais , Cumarínicos/química , Rutaceae/química , Anti-Inflamatórios/farmacologia , Extratos Vegetais/química , Óxido Nítrico , Estrutura Molecular
8.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 18-25, 2023.
Artigo em Chinês | WPRIM | ID: wpr-984579

RESUMO

ObjectiveTo explore the mechanism of Buyang Huanwutang in regulating macrophage polarization based on the Toll-like receptor 4 (TLR4) / nuclear factor-κB (NF-κB) / nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) pathway. MethodRAW264.7 macrophages were intervened with lipopolysaccharide (LPS) of different concentrations (0, 1.25, 2.5, 5, 10, 20, 40, and 80 mg·L-1) for 24 hours. Cell Counting Kit-8 (CCK-8) assay was used to determine the cell viability of RAW264.7 macrophages. The optimal concentration was chosen to establish an in vitro inflammation model induced by LPS. Cells were divided into a blank group (20% blank serum), a model group (20% blank serum + 10 mg·L-1 LPS), a model control group (20% FBS + 10 mg·L-1 LPS), low-, medium-, and high-dose (5%, 10%, and 20%) Buyang Huanwutang-containing serum groups, a high-dose (20%) Buyang Huanwutang combined with NLRP3 inhibitor MCC950 (50 μmol·L-1) group, a high-dose (20%) Buyang Huanwutang combined with reactive oxygen species (ROS) inhibitor NAC (10 μmol·L-1) group, and a high-dose (20%) Buyang Huanwutang combined with NF-κB inhibitor PDTC (10 μmol·L-1) group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of interleukin-1β (IL-1β), interleukin-18 (IL-18), and tumor necrosis factor-α (TNF-α) in RAW264.7 macrophages. Flow cytometry was employed to measure ROS levels in macrophages. Western blot was used to determine the protein expression of M1-type macrophage-related factors inducible nitric oxide synthase (iNOS) and TNF-α, M2-type macrophage-related factors arginase-1 (Arg-1) and interleukin-10 (IL-10), as well as the proteins in the TLR4/NF-κB/NLRP3 pathway. ResultCCK-8 results indicated that under 10 mg·L-1 LPS stimulation, RAW264.7 macrophages exhibited the highest cell viability (P<0.01). Compared with the blank group, the model group showed significantly increased levels of IL-1β, IL-18, and TNF-α (P<0.05,P<0.01), increased ROS expression (P<0.05,P<0.01), increased protein expression of M1-type macrophage factors iNOS and TNF-α (P<0.01), decreased protein expression of M2-type macrophage factors Arg-1 and IL-10 (P<0.05,P<0.01), and upregulated expression levels of TLR4, myeloid differentiation factor 88 (MyD88), phosphorylated inhibitor of NF-κB (p-IκB)/NF-κB inhibitor (IκB), phosphorylated NF-κB (p-NF-κB) p65/NF-κB p65, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and pro-Caspase-1 (P<0.05, P<0.01). Compared with the model group, all Buyang Huanwutang-treated groups and inhibitor groups significantly reduced levels of IL-1β, IL-18, and TNF-α (P<0.01), suppressed the expression of inflammatory factors in RAW264.7 macrophages, decreased cellular ROS expression levels (P<0.01), downregulated M1-type macrophages iNOS and TNF-α protein expression (P<0.01), upregulated M2-type macrophages Arg-1 and IL-10 protein expression (P<0.01), and lowered protein expression levels of TLR4, MyD88, p-IκB/IκB, p-NF-κB p65/NF-κB p65, NLRP3, ASC, and pro-Caspase-1 (P<0.05, P<0.01). ConclusionBuyang Huanwutang can improve macrophage inflammation, potentially by reducing macrophage ROS levels, inhibiting RAW264.7 macrophage polarization, and downregulating the protein expression levels of the TLR4/NF-κB/NLRP3 pathway.

9.
China Journal of Chinese Materia Medica ; (24): 4187-4200, 2023.
Artigo em Chinês | WPRIM | ID: wpr-1008615

RESUMO

This study aimed to explore the mechanism of Qilongtian Capsules in treating acute lung injury(ALI) based on network pharmacology prediction and in vitro experimental validation. Firstly, UPLC-Q-TOF-MS/MS was used to analyze the main chemical components of Qilongtian Capsules, and related databases were used to obtain its action targets and ALI disease targets. STRING database was used to build a protein-protein interaction(PPI) network. Metascape database was used to conduct enrichment analysis of Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG). AutoDock software was used to perform molecular docking verification on the main active components and key targets. Then, the RAW264.7 cells were stimulated with lipopolysaccharide(LPS) for in vitro experiments. Cell viability was measured by MTT and ROS level was measured by DCFH-DA. NO content was measured by Griess assay, and IL-1β, IL-6, and TNF-α mRNA expression was detected by RT-PCR. The predicted targets were preliminarily verified by investigating the effect of Qilongtian Capsules on downstream cytokines. Eighty-four compounds were identified by UPLC-Q-TOF-MS/MS. Through database retrieval, 44 active components with 589 target genes were screened out. There were 560 ALI disease targets, and 65 intersection targets. PPI network topology analysis revealed 10 core targets related to ALI, including STAT3, JUN, VEGFA, CASP3, and MMP9. KEGG enrichment analysis showed that Qilongtian Capsules mainly exerted an anti-ALI effect by regulating cancer pathway, AGE-RAGE, MAPK, and JAK-STAT signaling pathways. The results of molecular docking showed that the main active components in Qilongtian Capsules, including crenulatin, ginsenoside F_1, ginsenoside Rb_1, ginsenoside Rd, ginsenoside Rg_1, ginsenoside Rg_3, notoginsenoside Fe, notoginsenoside G, notoginsenoside R_1, notoginsenoside R_2, and notoginsenoside R_3, had good binding affinities with the corresponding protein targets STAT3, JUN, VEGFA, CASP3, and MMP9. Cellular experiments showed that Qilongtian Capsules at 0.1, 0.25, and 0.5 mg·mL~(-1) reduced the release of NO, while Qilongtian Capsules at 0.25 and 0.5 mg·mL~(-1) reduced ROS production, down-regulated mRNA expression of IL-1β, IL-6, TNF-α, and inhibited the inflammatory cascade. In summary, Qilongtian Capsules may exert therapeutic effects on ALI through multiple components and targets.


Assuntos
Humanos , Fator de Necrose Tumoral alfa , Ginsenosídeos , Caspase 3 , Metaloproteinase 9 da Matriz , Interleucina-6 , Simulação de Acoplamento Molecular , Farmacologia em Rede , Espécies Reativas de Oxigênio , Espectrometria de Massas em Tandem , Lesão Pulmonar Aguda/genética , Cápsulas , RNA Mensageiro , Medicamentos de Ervas Chinesas/farmacologia
10.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 106-112, 2023.
Artigo em Chinês | WPRIM | ID: wpr-972291

RESUMO

ObjectiveTo investigate the mechanism of Lycium barbarum polysaccharides (LBP) in promoting the activation of RAW264.7 macrophages. MethodRAW264.7 macrophages were stimulated with LBP at different concentrations (50, 100, 200 mg·L-1), and those stimulated with lipopolysaccharide (LPS) at 100 μg·L-1 and galactose (Gal) at 100 mg·L-1 as positive controls. After 24 h of LBP stimulation, the cell counting kit-8 (CCK-8) was used to detect the survival rate of RAW264.7 macrophages treated with LBP (0, 50, 100, 200, 400, 800 mg·L-1). The levels of interleukin-6 (IL-6) and interleukin-12 (IL-12) in cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). The protein and mRNA expression of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) in Toll-like receptor 4 (TLR4)/Toll-like receptor 2 (TLR2)/macrophage galactose-type lectin (MGL) pathway of RAW264.7 macrophages was detected by Real-time fluorescence-based quantitative polymerase chain reaction (Real-time PCR) and Western blot. ResultCCK-8 results showed that compared with the results in the blank group, the survival rate of RAW264.7 macrophages decreased in the 400, 800 mg·L-1 LBP groups (P<0.05). ELISA results showed that compared with the blank group, 50 mg·L-1 LBP could promote the secretion of IL-12 in RAW264.7 macrophages (P<0.01). Compared with the blank group, 100 mg·L-1 LBP and 200 mg·L-1 LBP could promote the secretion of IL-6 in RAW264.7 macrophages (P<0.05, P<0.01). Western blot results showed that compared with the blank group, the LBP groups (50, 100, 200 mg·L-1) enhanced protein expression levels of MAPK key molecules (p-p38 MAPK, p-ERK, p-NF-κB, and p-JNK) in TLR4, TLR2, and MGL pathways (P<0.05, P<0.01). Compared with the model group, the 200 mg·L-1 LBP group could promote the expression level of p-NF-κB protein in RAW264.7 macrophages (P<0.01). Real-time PCR results showed that compared with the blank group, the LBP groups (50, 100, and 200 mg·L-1) enhanced the mRNA expression levels of MAPK key molecules (p38 MAPK, ERK, NF-κB, and JNK) in TLR4 and TLR2 pathways (P<0.05, P<0.01). Compared with the model group, the 50 and 200 mg·L-1 LBP groups could promote the mRNA expression levels of JNK and ERK2 in RAW264.7 macrophages (P<0.05, P<0.01). ConclusionLBP can regulate the activation of RAW264.7 macrophages and participate in the immune response through the TLR2/TLR4/MGL pathway.

11.
Chinese journal of integrative medicine ; (12): 905-913, 2023.
Artigo em Inglês | WPRIM | ID: wpr-1010302

RESUMO

OBJECTIVE@#To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages.@*METHODS@#RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2-) radical and nitrite scavenging activity were also measured.@*RESULTS@#The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2- radical and nitrite scavenging activity.@*CONCLUSION@#EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.


Assuntos
Animais , Camundongos , Antioxidantes/farmacologia , Lipopolissacarídeos/farmacologia , Polygala , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Etanol/química , Interleucina-6/metabolismo , Anti-Inflamatórios/química , Espécies Reativas de Oxigênio/metabolismo , Ciclo-Oxigenase 2/metabolismo , Nitritos/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Superóxido Dismutase/metabolismo , RNA Mensageiro , Óxido Nítrico Sintase Tipo II/metabolismo
12.
Chinese Journal of Biochemistry and Molecular Biology ; (12): 91-96, 2022.
Artigo em Chinês | WPRIM | ID: wpr-1015742

RESUMO

Trimethylamine N-oxide (TMAO), a metabolite of intestinal flora, can promote Atherosclerosis (AS) in various ways. Current studies have found that it has a close relationship with plaque stability in clinical practice, but its molecular mechanism remains unclear at present. Extracellular matrix metalloproteinase inducers (CD147) / matrix metalloproteinases (MMPs) regulate a signal pathway highly related to plaque stability, which can promote plaque instability and lead to cardiovascular adverse events by weakening the thickness of the fibrous cap. Therefore, in this study, the mouse macrophageRAW264. 7 was stimulated by TMAO to establish a cell model to observe the effects on CD147, MMP2, and MMP9, and the CD147 gene silencing model was further constructed by using the siRNA transfection method to explore the interaction between CD147 and MMP2 and MMP9. Rt-qPCR and Western blotting results showed that there was no significant change in the gene expression level of CD147 in mouse macrophage RAW264. 7, but significantly increased in protein levels (P < 0. 05), while MMP2 andMMP9 were increased in mRNA and protein levels (P<0. 05). The expression of CD147, MMP2, andMMP9 was significantly inhibited in CD147 siRNA transfected cells (P<0. 05). In conclusion, TMAO significantly increases the expression of MMP2 and MMP9 in mouse macrophages RAW264. 7, and this effect may be partially realized through the CD147/ MMP pathway.

13.
Chinese Pharmacological Bulletin ; (12): 1340-1349, 2022.
Artigo em Chinês | WPRIM | ID: wpr-1014013

RESUMO

Aim To investigate the anti-inflammatory effects of Aesculetin from Viola tianshanica Maxim in LPS-stimulated RAW 264.7 cells and the underlying mechanism.Methods RAW 264.7 cells were divided into control group, model group( LPS), 0.16, 0.8, 4, 20 μmol·L-1 AESN groups( different concentrations of AESN + LPS)and positive control group(10 μmol·L-1 Indomethacin+LPS).LPS(1 mg·L-1)was used to stimulate RAW 264.7 cells for 24 h to establish inflammatory model.MTS assay was used to detemine cytotoxicity of Aesculetin in RAW 264.7 cells.Griess method was used to detect NO secretion in LPS-stimulated RAW 264.7 cells.ELISA was applied to determine the contents of TNF-α, IL-6 and IL-1β in cell culture supernatant.qRT-PCR was employed to detect the mRNA expression levels of TNF-α, IL-6, IL-1β and iNOS.Immunofluorescence assay was used to evaluated the protein expressions of iNOS, p-NF-κB p65, IκBα, p-p38 and p-ERK1/2.Enzyme assay was used to detect the inhibition activity of Aesculetin on cyclooxygenase 1/2(COX 1/2).Results Aesculetin significantly inhibited the secretion of inflammatory mediator NO, mRNA and protein expression of iNOS in LPS-induced RAW 264.7 at 0.16, 0.8, 4 and 20 μmol·L-1.The contents of TNF-α, IL-6 and IL-1β in supernatant significantly decreased, and the mRNA expression levels of TNF-α, IL-6 and IL-1β were also reduced by Aesculetin.Aesculetin also obviously inhibited the protein degradation of IκBα and inhibited the nuclear translocation of p-NF-κB p65, p-p38, p-ERK1/2.In addition, Aesculetin had significant inhibitory activities on COX-1 and COX-2, and the IC50 was 28.1 μmol·L-1, 2.3 μmol·L-1, respectively.Conclusions AESN has good anti-inflammatory effect, and its mechanism is closely related to the inhibition of NF-κB and MAPK signaling pathways

14.
Chinese Journal of Tissue Engineering Research ; (53): 3691-3699, 2022.
Artigo em Chinês | WPRIM | ID: wpr-930780

RESUMO

BACKGROUND: Interleukin-1 is an important pro-inflammatory cytokine that has been documented in the regulation of bone inflammation and bone remodeling. A previous study has demonstrated that interleukin-1α can induce apoptosis while inhibiting osteoblast differentiation in MC3T3-E1 cells. OBJECTIVE: To investigate the role and mechanism of interleukin-1α on osteoclast activation and bone loss in mice. METHODS: (1) Cell test: RAW264.7 cells were either treated with interleukin-1α alone or with receptor activator of nuclear factor-κB ligand (RANKL) for 1 and 4 days. Cell viability was tested by cell counting kit-8 assay. The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay. The mRNA and protein levels of osteoclast-specific genes and genes related to nuclear factor-κB pathway and Wnt/β-catenin pathway were tested by real-time fluorescence quantitative PCR, immunofluorescence staining or western blot. Bone marrow-derived macrophages were either treated with interleukin-1α alone or with RANKL and macrophage colony-stimulating factor for 7 days. The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay. The protein levels of osteoclast-specific genes were tested by western blot. (2) Animal test: Twenty-four male C57BL/6J mice (6-8 weeks old) were assigned into two groups at random: control group and test group. Mice were subsequently treated with interleukin-1α solution or PBS by intraperitoneal injection twice a week for 5 weeks. Bone tissues from the femurs were performed with micro-computed tomography analysis and hematoxylin-eosin staining, tartrate resistant acid phosphatase, and immunofluorescence analysis. RESULTS AND CONCLUSION: Cell test: Interleukin-1α alone significantly increased RAW264.7 cell proliferation, but stimulated cell differentiation into osteoclasts in combination with RANKL (P < 0.05). Interleukin-1α significantly increased the expression of osteoclast-related markers and the number of tartrate resistant acid phosphatase-positive multinuclear cells in RAW264.7 cells and bone marrow-derived macrophages in the existence of RANKL or RANKL+macrophage colony-stimulating factor (both P < 0.05). Interleukin-1α was found to significantly enhance the nuclear factor-κB and Wnt/beta-catenin signaling in RAW264.7 cells (P < 0.05). Blocking of nuclear factor-κB or Wnt3 signaling not only reversed the activation of nuclear factor-κB and Wnt3 signaling but also weakened the enhanced expression of osteoclast-specific genes induced by interleukin-1α in RAW264.7 cells (P < 0.05). Animal test: interleukin-1α induced bone loss in mice while also upregulating the expression of osteoclast-specific markers, RANK, TRAF6 and p65, and Wnt3 in vivo (P < 0.05). The findings indicate that interleukin-1α can induce osteoclast activation and bone loss by promoting the nuclear factor-κB and Wnt signaling pathways.

15.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 36-42, 2022.
Artigo em Chinês | WPRIM | ID: wpr-940349

RESUMO

ObjectiveTo explore the effect and mechanism of Xiaojindan extract (XJD) on macrophage polarization. MethodLipopolysaccharide (LPS) and interleukin-4 (IL-4) were used to induce M1 and M2 polarization of RAW264.7 cells. The influence of 10-80 mg·L-1 XJD on cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Nitric oxide (NO) and interleukin-6 (IL-6) release was explored by Griess assay and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA expression of M1 and M2 macrophage markers was measured by real-time quantitative polymerase chain reaction (Real-time PCR), and the CD206+ expression was determined by flow cytometry. The activation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway was analyzed by western blot. Result10-80 mg·L-1 XJD showed no marked cytotoxicity in LPS (0.5 mg·L-1)- or IL-4 (20 μg·L-1)-induced RAW264.7 cells. Compared with the control group, LPS significantly promoted the expression of M1 macrophage markers (P<0.01), including increased NO and IL-6 release (P<0.01) and upregulated mRNA expression of interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) (P<0.01). Compared with LPS-induced group, 20-80 mg·L-1 XJD decreased the release of NO and IL-6 in a dose-dependent manner (P<0.01), and similarly 10-80 mg·L-1 XJD suppressed the mRNA expression of IL-1β, iNOS, COX-2 and TNF-α (P<0.01). Compared with the control group, IL-4 obviously increased the expression of M2 macrophage markers (P<0.01), including increased CD206+ cell population and upregulated mRNA expression of arginine-1 (Arg-1), interleukin-10 (IL-10), interleukin-13 (IL-13) and transforming growth factor-β1 (TGF-β1). Compared with IL-4-induced group, 10-80 mg·L-1 XJD dose-dependently decreased CD206+ cell population (P<0.01) and inhibited the mRNA expression of Arg-1, IL-10, IL-13 and TGF-β1 (P<0.01). Western blot showed that XJD significantly downregulated the activation of PI3K/Akt pathway as compared to LPS- and IL-4-induced groups (P<0.05, P<0.01). ConclusionXJD significantly inhibited the macrophage polarization in the LPS- and IL-4-induced RAW264.7 cells by targeting PI3K/Akt pathway.

16.
Journal of Zhejiang University. Science. B ; (12): 230-240, 2022.
Artigo em Inglês | WPRIM | ID: wpr-929054

RESUMO

Marine fungi are important members of the marine microbiome, which have been paid growing attention by scientists in recent years. The secondary metabolites of marine fungi have been reported to contain rich and diverse compounds with novel structures (Chen et al., 2019). Aspergillus terreus, the higher level marine fungus of the Aspergillus genus (family of Trichocomaceae, order of Eurotiales, class of Eurotiomycetes, phylum of Ascomycota), is widely distributed in both sea and land. In our previous study, the coral-derived A. terreus strain C23-3 exhibited potential in producing other biologically active (with antioxidant, acetylcholinesterase inhibition, and anti-inflammatory activity) compounds like arylbutyrolactones, territrems, and isoflavones, and high sensitivity to the chemical regulation of secondary metabolism (Yang et al., 2019, 2020; Nie et al., 2020; Ma et al., 2021). Moreover, we have isolated two different benzaldehydes, including a benzaldehyde with a novel structure, from A. terreus C23-3 which was derived from Pectinia paeonia of Xuwen, Zhanjiang City, Guangdong Province, China.


Assuntos
Animais , Camundongos , Acetilcolinesterase/metabolismo , Antozoários/microbiologia , Anti-Inflamatórios/farmacologia , Aspergillus/química , Benzaldeídos/farmacologia , Transdução de Sinais
17.
Chinese Journal of Microbiology and Immunology ; (12): 705-713, 2022.
Artigo em Chinês | WPRIM | ID: wpr-958246

RESUMO

Objective:To investigate the regulatory effects of mitofusin 1 (MFN1) on lipopolysaccharide (LPS)-induced Raw264.7 mouse macrophages pyroptosis and to provide reference for further study on the prevention of inflammation and fibrosis caused by macrophage dysfunction.Methods:Raw264.7 mouse macrophages were cultured in vitro and used to construct a model of LPS-induced pyroptosis. CCK-8 staining, PI staining, LDH release assay and Western blot were used to verify the Raw264.7 pyroptosis induced by LPS. MFN1 expression was detected by Western blot. DCFH-DA probe was used to detect the synthesis of total reactive oxygen species (ROS); Mito-SOX was used to detect mitochondrial ROS; JC-1 mitochondrial membrane potential was detected by fluorescence probe to reflect mitochondrial damage. Based on Ubibrowser database, it was predicted that MFN1 could bind to a variety of E3 ubiquitin ligases. Then, immunofluorescence and co-immunoprecipitation (CO-IP) were used to analyze MFN1 ubiquitination. An overexpression plasmid for MFN1 was constructed and transfected into Raw264.7 cells to detect the changes in pyroptosis and mitochondrial function. Results:LPS could induce the pyroptosis of Raw264.7 cells and mitochondrial dysfunction. MFN1 expression was decreased after LPS stimulation. Ubiquitinated MFN1 was detected by CO-IP. Ubiquitination inhibitor MG-132 inhibited LPS-induced expression of pyroptosis-related proteins including NLRP3, Pro-caspase-1, Caspase-1, IL-1β and IL-18 and improved mitochondrial function. MFN1 overexpression relieved the mitochondrial dysfunction and pyroptosis of Raw264.7 cells induced by LPS.Conclusions:The ubiquitination of MFN1 induced by LPS was involved in mitochondrial dysfunction and macrophage pyroptosis, suggesting that MFN1 was a potential target for the treatment of macrophage-induced inflammation and related diseases.

18.
Chinese Pharmacological Bulletin ; (12): 1614-1619, 2021.
Artigo em Chinês | WPRIM | ID: wpr-1014509

RESUMO

Aim To explore the anti-inflammatory effect of taurolithocholic acid (TLCA) through network pharmacology-based analyses, to verify with in vitro macrophage study and to reveal the possible mechanisms. Methods The potential targets of TLCA were acquired from public database, and then the protein-protein interaction (PPI) networks against inflammation were constructed and visualized by using Cytoscape. Gene ontology (GO) analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed. The binding activity of TLCA and its target (TGR5) was evaluated through molecular docking analysis. Lastly, the results of the network analysis were confirmed by lipopolysaccharide and interferon-γ induced RAW264.7 cells. Results There were 87 anti-inflammatory potential targets were screened. GO analysis revealed gene functions were mainly involved in regulation of inflammatory response, membrane raft and protein tyrosine kinase. The results of KEGG pathway analysis suggested that PI3K-Akt signaling pathway, human cytomegalovirus infection might be the critical pathways of TLCA against inflammation. The results of in vitro experiments showed that TLCA decreased the LPS and IFN-γ induced inflammatory response in RAW 264.7 macrophages. Furthermore, the expression of TGR5 protein increased after TLCA treatment. Conclusions The potential therapeutic targets of TLCA against inflammation are revealed through network pharmacology analysis. Our results indicate that TLCA might regulate key inflammatory markers through the membrane receptor TGR5.

19.
Chinese Pharmacological Bulletin ; (12): 1092-1098, 2021.
Artigo em Chinês | WPRIM | ID: wpr-1014486

RESUMO

Aim To investigate the effects of Kudino- side D on lipid accumulation induced by oxidized low density lipoprotein ( ox-LDL) and inflammation induced by lipopolysaccharide ( LPS ) in RAW264.7 cells.Methods Foam cells were established by incubating the RAW264.7 cells with ox-LDL.The concentration of lipid droplets in the cells was observed by oil red staining, and the level of total cholesterol (TC) in cells was measured by enzyme method.The gene and protein expressions of scavenger receptors CD36 and SR-A1, ATP binding cassette transporters A1 and Gl ( ABCA1 and ABCGI) were detected by RT-qPCR and Western blot, respectively.The expressions of inter- leukin-6 (IL-6), interleukin-1 (3 (IL-ip), monocyte chemoattractant protein-1 (MCP-1 ) and tumor necrosis factor-a (TNF-a) were detected by ELISA and RT-qPCR.The protein expressions of mTOR and p-mTOR were detected by Western blot.Results Compared with model group, the high dose of Kudinoside D decreased the content of TC and down-regulated the gene and protein expression of SR-A induced by ox-LDL.Meanwhile Kudinoside D also decreased the levels of IL-ip and MCP-1 and down-regulated the protein expression of p-mTOR induced by LPS.Conclusions Kudinoside D may reduce the intracellular TC content by down-regulating the gene and protein expression of SR-A1.Kudinoside D may play an anti-inflammatory role through mTOR pathway.

20.
Chinese Pharmacological Bulletin ; (12): 233-239, 2021.
Artigo em Chinês | WPRIM | ID: wpr-1014322

RESUMO

Aim To design and synthesizea new pyrazolo[4,3-d] pyrimidine derivative for the purpose of developing novel anti-inflammatory agents, and to revealthe possible anti-inflammatory mechanisms of thenew compound using preliminary studies. Methods The changes of the cell morphology were investigated via the microscope. The influence of title compound on the NO production of the L P S - stimulated RAW264. 7 cells was measured by Griess assay. The gene relative transcription levels of TNF-a, IL - 6 and IL-lß were evaluated by qRT-PCR. COX-2 expression was measured by qRT-PCR and western blot. NF-KB/NLRP3 inflammasome signaling pathway were investigated by Western blot. The changes of lung tissue were observed by HE staining in vivo experiments of mice. Results Preliminary mechanism researches revealed that the ti - tle compound could inhibit nitric oxide (NO) generation as well as the expression of COX-2, TNF-a, IL - 6, and IL - lβ by NF-KB/NLRP3 inflammasome signaling pathway in RAW264. 7 cells. Furthermore, it could simultaneously improve the morphology of the cells and relieve acute lung injury in mice. Conclusions The new pyrazolo [4, 3-α] pyrimidine derivative has anti-inflammatory activity through NF-KB/NLRP3 inflammasome signaling pathway.

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