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BACKGROUND:Previous studies have shown that puerarin can inhibit the differentiation of osteoclasts,and the expression of Notch signaling pathway-related proteins such as Notch1,HES1,and Jagged1 is decreased.However,the specific mechanism of the Notch1 signaling pathway for the inhibition of osteoclast differentiation by puerarin is not clear. OBJECTIVE:To explore the effect of Notch signaling pathway on puerarin inhibiting the differentiation of mouse macrophage Raw264.7 into osteoclasts. METHODS:Raw264.7 cells were divided into seven groups for intervention culture.Blank control group was cultured in high-sugar DMEM medium;the osteoclast induction group was cultured in osteoclast induction medium;the puerarin intervention group was cultured with 50 μmol/L puerarin at the same time of osteoclast induction;Notch1 siRNA control group,Notch1 siRNA group,Notch1 overexpression control group and Notch1 overexpression group were transfected with Notch1 siRNA control sequence,Notch1 siRNA,Notch1 overexpression control plasmid and Notch1 overexpression plasmid,respectively,and then cultured with osteoclast induction medium and puerarin.The number and size of osteoclasts were observed by tartrate-resistant acid phosphatase staining,the skeleton formation of osteoclasts was observed by F-actin staining,and the gene expression level of osteoclast formation markers was detected by RT-PCR. RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase staining results showed that puerarin intervention could inhibit the generation of osteoclasts,Notch1 silencing could further reduce the number of osteoclasts,while the number of osteoclasts in the osteoclast-induced group increased significantly after Notch1 overexpression.The results of F-actin showed that Raw264.7 cells could form a well-defined F-actin ring after osteoclast induction.Puerarin intervention would inhibit the formation of cytoskeleton,and Notch1 silencing could aggravate the inhibitory effect of cytoskeleton formation,while Notch1 overexpression could alleviate this inhibitory effect of puerarin.RT-PCR results showed that puerarin could inhibit the mRNA expression levels of tartrate-resistant acid phosphatase,Cathepsin K and c-Fos,the expression of the above-mentioned three factors decreased significantly after Notch1 gene silencing,and Notch1 overexpression could upregulate the expression of these factors.These finding indicate that puerarin inhibits the differentiation of Raw264.7 cells into osteoclasts through the Notch signaling pathway.
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Objective To explore the mechanism of Nuanxinkang Powder(aka.NXK,composed of Ginseng Radix et Rhizoma Rubra and Ilex Pubescens Radix)on improving ventricular remodeling in post-infarction mice based on the"metabolic-inflammatory"network regulating macrophage polarization.Methods ①Thirty C57BL/6J male mice were randomly divided into three groups:sham-operation group,model group,and NXK group(1.65 g·kg-1),with 10 mice in each group;the mouse model of myocardial infarction was replicated using left anterior descending coronary artery ligation;and the drug was administered by gavage once a day for 4 consecutive weeks.Masson staining was used to detect collagen deposition in myocardial tissue;ultrasound was used to detect cardiac function in mice:left ventricular ejection fraction(LVEF),left ventricular anterior wall thickness at end-systole(LVAWS)and left ventricular anterior wall thickness at end-diastole(LVAWD);flow cytometry was used to detect distribution of cardiac macrophages in mice;qPCR was used to detect mRNA expressions of lactate dehydrogenase A(LDHA),carnitine palmitoyltransferase 1(CPT-1),glucose transport protein 4(GLUT4),isocitrate dehydrogenase(IDH),and succinate dehydrogenase(SDHa)in heart tissue.②NXK was given 1.15 g·kg-1 NXK suspension to rats by gavage twice a day for 5 consecutive days to prepare NXK-containing serum.Lipopolysaccharide(LPS)-induced RAW 264.7 cells were used to construct a pro-inflammatory macrophage model.The cells were grouped into the following groups:blank serum control group(medium containing 5%blank serum+5%fetal bovine serum),NXK drug-containing serum group(medium containing 5%NXK drug-containing serum+5%fetal bovine serum),lipopolysaccharide group(medium containing 5%blank serum+5%fetal bovine serum+200 μg·mL-1 lipopolysaccharide),NXK drug-containing serum+ lipopolysaccharide group(medium containing 5%NXK drug-containing serum+5%fetal bovine serum+200 μ g·mL-1 lipopolysaccharide),all the groups were intervened for 16 hours.Glycolysis stress test was used to detect the level of glycolysis in RAW 264.7 cells;qPCR was used to detect the mRNA expression of mitochondrial pyruvate carrier(MPC1)in RAW 264.7 cells;and MitoSox Red fluorescent staining was used to detect the level of oxidative stress damage in mitochondria of RAW 264.7 cells.Results ①Compared with the sham-operation group,the blue-stained area of cardiac collagen fibres in mice of the model group was significantly increased,accompanied by thinning of the ventricular wall and enlargement of the left ventricular cavity;cardiac function indexes,such as LVEF,LVAWS,LVAWD,etc.,were all significantly reduced(P＜0.01,P＜0.001);the mRNA expressions of LDHA and CPT-1 were significantly up-regulated in the cardiac tissues of mice(P＜0.05),and the mRNA expressions of GLUT4,IDH and SDHa were significantly down-regulated(P＜0.05,P＜0.01),and CD86 staining positive cell was significantly increased(P＜0.001).Compared with the model group,mice in the NXK group showed a significant decrease in cardiac collagen fiber deposition and an increase in the thickness of the ventricular wall;cardiac function indexes such as LVEF,LVAWS and LVAWD were significantly increased(P＜0.05,P＜0.01,P＜0.001);and the mRNA expressions of LDHA and CPT-1 in the cardiac tissues of the mice were significantly down-regulated(P＜0.01,P＜0.001),mRNA expressions of GLUT4,SDHa and IDH were significantly up-regulated(P＜0.01),and the number of CD86 positive cells was significantly reduced(P＜0.001).②Compared with the blank serum control group,the cytosolic glycolysis level and ROS level of macrophages in the NXK serum-containing group did not change significantly(P＞0.05),whereas the glycolysis level and ROS level of macrophages in the lipopolysaccharide group were significantly increased(P＜0.01),and the mRNA expression of MPC1 was significantly down-regulated(P＜0.001).Compared with the lipopolysaccharide group,the macrophage glycolysis level and ROS level were significantly reduced in the NXK serum-containing + lipopolysaccharide group(P＜0.05,P＜0.01),and mRNA expression of MPC1 was significantly up-regulated(P＜0.001).Conclusion NXK can reduce myocardial fibrosis and ventricular remodeling after myocardial infarction and improve cardiac function in mice,and its mechanism may be related to the down-regulation of mRNA expression of LDHA in cardiac tissues,the up-regulation of mRNA expression of GLUT4,the improvement of cardiac glucose uptake after myocardial infarction,the inhibition of pro-inflammatory macrophage glycolysis,the increase in the expressions of SDHa and IDH to alleviate the accumulation of succinate and citrate,and the reduction of reactive oxygen species(ROS)generation,thereby reducing pro-inflammatory macrophage hyperpolarisation.
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Objective To study the effect and its mechanism of hederagenin(hed)on dextran sulfate sodium(DSS)-induced ulcerative colitis(UC)in mice.Methods(1)In vitro experiments:after treating RAW264.7 cells with different concentrations(0,2.5,5,10,20,40 μmol·L-1)of hed for 24 hours,the cell survival rate was detected by MTT assay.RAW264.7 cells were divided into:blank group,lipopolysaccharide(LPS)group(1 μg·L-1),LPS+2.5 μmol·L-1 hed group,LPS+5 μmol·L-1 hed group and LPS+10 μmol·L-1 hed group;an in vitro cellular inflammation model was established using LPS intervention for 24 hours and co-incubated with hed for 24 hours.The levels of interleukin 1β(IL-1β),IL-6 and tumor necrosis factor α(TNF-α)in the cell supernatant were determined by ELISA;the expression levels of TLR4/NF-κB pathway-related proteins in the cells were detected by Western Blot.(2)In vivo experiments:C57BL/6 mice were randomly divided into a blank group,a model group,a Salazosulfapyridine group(200 mg·kg-1),and an hed low-,medium-,and high-dosage groups(12.5,25,and 50 mg·kg-1),with 5 mice in each group.Mice were induced to establish UC model by drinking 3%DSS solution freely for 7 days.The UC model was then established by gavage once a day for 7 days.At the end of the administration,the Disease Activity Index(DAI)was evaluated;pathological changes in the colonic tissues of mice were observed by HE staining;the levels of IL-1β,IL-6,and TNF-α in the colonic tissue were measured by ELISA;and the expression levels of proteins related to the TLR4/NF-κB pathway in the colonic tissue were detected by Western Blot.Results(1)In vitro experiments:compared with the blank group(0 μ mol·L-1 group),there was no significant change in the cell survival rate in the 2.5-10 μmol·L-1 hed group(P＞0.05),and there was no significant toxicity effect on RAW264.7 cells.Compared with the blank group,the expression levels of IL-1β,IL-6,and TNF-α in RAW264.7 cells in the LPS group were significantly increased(P＜0.01);and the protein expression levels of TLR4 and p-NF-κ B/NF-κ B were significantly increased(P＜0.01).Compared with the LPS group,the expression levels of IL-1β and TNF-α in RAW264.7 cells in the hed 2.5,5,and 10 μmol·L-1 concentration groups were significantly decreased(P＜0.05,P＜0.01),and the protein expression levels of TLR4,p-NF-κB/NF-κB were significantly decreased(P＜0.05,P＜0.01);the IL-6 expression level of RAW264.7 cells in the hed 5 and 10 μmol·L-1 concentration groups was significantly reduced(P＜0.05,P＜0.01).(2)In vivo experiments:compared with the blank group,the body mass of mice in the model group was consistently reduced(P＜0.01),the DAI score was significantly elevated(P＜0.01),and the length of the colon was significantly shortened(P＜0.01);the colonic tissue showed obvious epithelial cell damage,and the histopathological scores were significantly elevated(P＜0.01);and the expression levels of the pro-inflammatory cytokines IL-1β,IL-6 and TNF-α were significantly increased(P＜0.01);protein expression levels of TLR4 and p-NF-κB/NF-κB were significantly increased(P＜0.01)in colon tissue.Compared with the model group,the body mass of mice in the low-,medium-and high-dose groups of hed were significantly increased(P＜0.05,P＜0.01),the DAI score was significantly decreased(P＜0.05,P＜0.01),the pathological damage of colon tissue improved to different degrees,and the protein expression levels of TLR4,p-NF-κB/NF-κB in the colonic tissue were significantly decreased(P＜0.05,P＜0.01);the colon length of mice in the medium-and high-dose groups of hed were significantly increased(P＜0.05,P＜0.01),and the expression levels and histopathological scores of IL-1β,IL-6,and TNF-α in colon tissue were significantly reduced(P＜0.05,P＜0.01).Conclusion Hed were able to effectively ameliorate colonic histopathological injury and reduce the levels of inflammatory factors in DSS-induced UC mice,and their mechanism of action may be related to the inhibition of the TLR4/NF-κB pathway.
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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.
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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
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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.
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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.
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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.
Sujet(s)
Animaux , Souris , Antioxydants/pharmacologie , Lipopolysaccharides/pharmacologie , Polygala , Facteur de transcription RelA/métabolisme , Facteur de nécrose tumorale alpha/métabolisme , Éthanol/composition chimique , Interleukine-6/métabolisme , Anti-inflammatoires/composition chimique , Espèces réactives de l'oxygène/métabolisme , Cyclooxygenase 2/métabolisme , Nitrites/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Monoxyde d'azote/métabolisme , Superoxide dismutase/métabolisme , ARN messager , Nitric oxide synthase type II/métabolismeRÉSUMÉ
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.
Sujet(s)
Souris , Animaux , Coumarines/composition chimique , Rutaceae/composition chimique , Anti-inflammatoires/pharmacologie , Extraits de plantes/composition chimique , Monoxyde d'azote , Structure moléculaireRÉSUMÉ
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.
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Objective:To investigate the anti-inflammatory effects of water extract of the<italic> Iris halophila</italic> root on lipopolysaccharide（LPS） stimulated RAW264.7 cells and analyze its chemical constituents. Method:The supernatant of YWG prepared by water extraction and alcohol precipitation was separated by AB-8 macroporous adsorption resin column chromatography to obtain ethanol eluates with different concentrations （YWG，YWG-0%，YWG-20%，YWG-40%，and YWG-60%）. Cell counting kit-8（CCK-8） assay was used to determine the effects of YWG-0%，YWG-20%，YWG-40%，and YWG-60% on the viability of RAW264.7 cells. Griess assay was employed to detect the nitric oxide （NO） level in LPS-stimulated RAW264.7 cells. The release of tumor necrosis factor（TNF）-<italic>α</italic>，interleukin（IL）-6，IL-10，and IL-1<italic>β</italic> was detected by enzyme-linked immunosorbent assay（ELISA）. YWG and the elution site with the most robust anti-inflammatory activity were identified and compared by ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry （UHPLC-Q-TOF-MS/MS）. Result:Ethanol eluates with different concentrations inhibited the release of NO，TNF-α，IL-1<italic>β</italic>， and IL-6 in the supernatant of LPS induced RAW264.7 cells （<italic>P<</italic>0.05），and promoted the release of IL-10 （<italic>P<</italic>0.05）. YWG-60% displayed a highly significant effect （<italic>P</italic><0.01）. A total of 127 constituents were detected from the comparison of YWG and YWG-60% by UHPLC-Q-TOF-MS/MS in the positive and negative ion modes，including 61 flavonoids. YWG-60% contained 25 flavonoids with elevated content as compared with YWG. Conclusion:YWG-60% showed potent anti-inflammatory effect，and the effective anti-inflammatory constituents were presumedly flavonoids. The findings of this study are expected to provide a scientific theoretical basis for the basic research on the medicinal effect of the water extract of YWG.
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Objective To study the immunomodulatory effect of polysaccharides (CRPS25-Ⅱ) derived from Chroogomphus rutilus on mouse mononuclear macrophages, RAW264.7 cells. Methods RAW264.7 cells were resuspended and cultured, cell suspension was prepared. The blank control group and CRPS25-Ⅱ groups with different mass concentrations (1, 20, 40, 80 and 160 μg/ml) were set up. MTT assay was used to determine the cytotoxicity of CRPS25-Ⅱ on RAW264.7 cells. RT-PCR was used to detect the effects of CRPS25-Ⅱ on the secretion of immune regulatory factors IL-6 and TNF-α from RAW264.7 cells. Western blot was used to detect the effects of CRPS25-Ⅱ on the expression of p-P65 protein in NF-κB pathway of RAW264.7 cells. Results The results showed that CRPS25-Ⅱ (1−160 μg/ml) had no obvious cytotoxicity. CRPS25-Ⅱ (1−160 μg/ml) increased the secretion of cytokines, and thus promoted the mRNA expression of IL-6 and TNF-α. CRPS25-Ⅱ increased the phosphorylation of p-P65 protein and activated the NF-κB signaling pathway, and thus promoted the immune regulation of cells. CRPS25-Ⅱ (1−160 μg/ml) could increase the p-P65 protein, and the promoting effects of CRPS25-Ⅱshowed an upward trend in the concentration range of 1−40 μg/ml and gradually weakened in the concentration range of 40−160 μg/ml. Conclusion Polysaccharides derived from chroogomphus rutilus had no cytotoxicity to mouse macrophages, and could promote the secretion of inflammatory factors IL-6 and TNF-α and activate the NF-κB signaling pathway, thus playing an immunomodulatory role.
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Objective: To investigate whether the ethanol extract of Chondracanthus tenellus (Harvey) Hommersand, a type of red algae, could exhibit anti-inflammatory potential in lipopolysaccharide (LPS)-stimulated macrophages. Methods: The ethanol extract of Chondracanthus tenellus was applied to 100 ng/mL LPS-stimulated RAW 264.7 cells, and cell viability, phagocytic ability, levels of pro-inflammatory factors, and the production of reactive oxygen species were measured. To identify the underlying mechanism of the ethanol extract of Chondracanthus tenellus, the expression of inflammation-regulated genes was estimated. Results: The ethanol extract of Chondracanthus tenellus had no cytotoxic effect at concentrations below 300 μg/mL, and reduced the LPS-induced production of inflammatory mediators including nitric oxide (NO) and prostaglandin E 2. Furthermore, the extract markedly suppressed the expression of inducible NO synthase and cyclooxygenase-2, as well as the production of reactive oxygen species. The LPS-induced up-regulation of pro-inflammatory cytokines was attenuated by treatment with the ethanol extract of Chondracanthus tenellus, reducing their extracellular secretion. The Chondracanthus tenellus extract also inhibited LPS-mediated activation of nuclear factor-kappa B (NF-κB). In addition, the phosphorylation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3 kinase (PI3K)/Akt was markedly increased by LPS, which was significantly abolished by the Chondracanthus tenellus extract. Conclusions: Our findings indicate that the ethanol extract of Chondracanthus tenellus exhibited potential anti-inflammatory and antioxidant effects through downregulating the NF-κB, MAPKs, and PI3K/Akt signaling pathways in LPS stimulated RAW 264.7 macrophages.
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OBJECTIVE@#To demonstrate the anti-inflammatory activity of Brassica napus L. hydrosols (BNH) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.@*METHODS@#Composition analysis of BNH was conducted via gas chromatography-mass spectrometry after BNH were extracted. The nitric oxide (NO) production was measured using the Griess assay. Prostaglandin E@*RESULTS@#Compared with LPS-stimulated cells, BNH markedly decreased the generation of NO and PGE@*CONCLUSION@#The anti-inflammatory activities of BNH were mediated via blockage of the NF-κB signaling pathways in LPS-stimulated RAW 264.7 cells.
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Aim To establishan inflammatory model of mouse monocyte macrophages ( RAW264. 7) using li-popolysaccharide (LPS), and to investigate the antiinflammatory activity and mechanism of tanshinone II-A (Tan IIA). Methods Cell viability was determined by CCK-8 method. Cell migration was detected by Tr-answell apparatus. TNF-α, IL-6, IL-1 p, MCP-1 content in cell supernatant was analyzed using ELISA method. The protein expression of MMP-2, MMP-9, TLR4, κB-α, p-κB-α, NFκB and p-NFκB in RAW264.7 cells was investigated by Western blot. Results Tan IIA significantly inhibited .the secretion of TNF-α, IL-6, IL-1β and MCP-1 in LPS induced RAW264.7 cell culture medium , and significantly down-regulated the expression of matrix metalloprotein-ase-2 (MMP-2), MMP-9, TLR4, p-κB-α and p-NFκB , inhibited IκB-α phosphorylation and NFκB entry into the nucleus and activation. Conclusion Tan IIA can inhibit the release of inflammatory factors through the regulation of TLR4/IκB-α/NFκB signaling pathway.
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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.
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Objective:To explore the effect of anemarrhena asphodeloside BⅡ (TBⅡ) on the expressions of nuclear transcription factor-κB receptor activator factor ligand （RANKL）, RANK and C-FOS genes during osteoclast differentiation. Method:Molecular docking software LeDock was used to score the docking of TBⅡ with RANKL, RANK and C-FOS. RAW264.7 was treated with soluble RANKL（sRANKL） and divided into control group, sRANKL group (model group), Icariin (Ica) group, low-dose TBIⅡ group (2 μmol·L-1), medium-dose TBⅡ group (4 μmol·L-1), and high-dose TBⅡ group (8 μmol·L-1). The corresponding kit was used to detect iconic enzyme (TRAP) of osteoclast differentiation. Total RNA was extracted by trizol method, Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect the expressions of C-FOS, upstream RANKL/RANK and downstream nuclear factor of activated T-cells cytoplasmic 1 (NFATC1), and osteoprotegerin OPG. Result:The molecular docking score were -11.86, -11.38, -12.34 kcal·mol-1, and there might be multiple binding sites between TBII as well as RANKL, RANK and C-FOS. Compared with the control group, the content of TRAP in model group increased significantly (P<0.01), and compared with model group, the content of TRAP in each administration group decreased significantly (P<0.01), and TBⅡ decreased the content of TRAP in a dose-dependent manner. Compared with the control group, the expressions of RANKL, RANK, C-FOS and NFATC1 increased (P<0.01), whereas the expression of OPG decreased (P<0.01) in model group. Compared with model group, the expressions of RANKL, RANK, C-FOS and NFATC1 decreased (P<0.01), while the expression of OPG increased (P<0.01) in each administration group. Conclusion:TBⅡ may inhibit the differentiation of osteoclast precursors into osteoclasts, inhibit osteoclast activity, reduce bone resorption and improve osteoporosis by regulating RANKL/RANK/C-FOS signal pathway.
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Aim To investigate the effect of genistein (GEN) on apoptosis of RAW264. 7 cells activated by lipopolysaccharide (LPS) and the possible pharmacological mechanisms. Methods RAW264. 7 cells and TIPE 2-over expression cells were preincubated with GEN for 2 h, then incubated with LPS for 24 h. CCK 8 kit was used to detect cell viability. Annexin V-FITC/PI kit was used to detect cell apoptotic rate. qRT-PCR was used to detect the level of TNF-a, IL-6, caspase-8, caspase-3 and TIPE 2 mRNA. Western blot was used to detect the expression of iNOS, COX-2, caspase-8, caspase-3, TIPE 2, Akt and p-Akt. Results LPS increased the synthesis of TNF-a, IL-6, iNOS and COX-2 in RAW264. 7 cells. GEN inhibited the activity, increased the apoptotic rate and the level of caspase-8, caspase-3 and TIPE 2 of LPS-activated RAW264. 7 cells. TIPE 2-over expression up-regulated the level of caspase-8, caspase-3 and reduced the expression of p-Akt, which were further enhanced by GEN in activated macrophage. Conclusions Genistein may promote the apoptosis of LPS-activated RAW264. 7 cells through inhibiting Akt activities by up-regulating TIPE 2 and activating the exogenous apoptotic pathway.
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OBJECTIVE：To investigate the intervention effect of Shenfu i njection（SFI）on the nuclear translocation of high mobility group box 1（HMGB1） in lipopolysaccharide （LPS）-induced RAW 264.7 cells. METHODS ： Using LPS-induced RAW264.7 cells as objects ，the histone deacetylase inhibitor RGFP 966 as positive control ，CCK-8 assay was used to screen drug dosage，and the effects of low ，medium and high doses （3，6，12 μL/mL）of SFI on HMGB 1 nuclear translocation in RAW 264.7 cells were observed by immunofluorescence method ；mRNA expression of HMGB 1 in RAW 264.7 cells were detected by real time fluorescent PCR. Western blotting assay was used to determine protein expression of HMGB 1 and Toll-like receptor 4（TLR4）；the expression of HMGB 1 were compared between nucleus and cytoplasm. The levels of HMGB 1，IL-1β and TNF-α in supernatant of cells were detected by ELISA. RESULTS ：In blank control group ，HMGB1 was mainly located in the nucleus ；after LPS induction， HMGB1 migrated from nucleus to cytoplasm. Compared with blank control group ， mRNA and protein （No.81760738） expression of HMGB 1， protein expression of TLR 4 in RAW264.7 cells as well as the levels of HMGB 1，IL-1β and TNF-α in supernatant of cells were increased significantly in LPS group （P＜0.01）. The protein expression of HMGB 1 was decreased significantly in nucleus while was in creased significantly in cytoplasm （P＜0.01）. After SFI treatment ，the nuclear translocation and secretion of HMGB 1 were inhibited in different degrees ；compared with LPS group ，mRNA and protein expression of HMGB 1 in administration groups ，protein expression of TLR 4 in RAW 264.7 cells of positive control group ，SFI medium- and high-dose groups as well as the levels of HMGB 1，IL-1β and TNF-α in supernatant of cells in administration groups were decreased significantly （P＜0.01）. In positive control group ，SFI medium- and high-dose groups ，the protein expressions of HMGB1 in nucleus were increased significantly ，while protein expressions of HMGB 1 in cytoplasm were decreased significantly （P＜0.01）. CONCLUSIONS ：SFI may inhibit the nuclear translocation and secretion of HMGB 1 in RAW 264.7 cells，thus avoiding the activation of inflammatory pathways and the production of inflammatory factors ，so as to reduce the inflammatory response induced by LPS.
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Objective: New type of nano-carbon dots were found after pyrolysis of human hair using motor oil as a dispersant and the biological effect of these carbon dots was evaluated by animal experiments. Methods: High-temperature pyrolysis was used to carbonize human hair and motor oil, and the carbonized products were extracted, filtered, and dialyzed to obtain a new type of water-soluble substance, carbon dots, named JYRF-CDs. JYRF-CDs were characterized using transmission electron microscopy (TEM) and high-resolution TEM, as well as ultraviolet-visible, fourier transform infrared, fluorescence spectroscopy and X-ray photoelectron spectroscopy (XPS). CCK-8 toxicity test using RAW264.7 cells was used to evaluate the safety of JYRF-CDs and the biological effects of the JYRF-CDs were evaluated by mouse ear swelling experiments and mouse acetate writhing experiments. Results: These JYRF-CDs were nearly spherical and well separated from each other, with a size distribution range of 1.8-3.6 nm, the CDs had a lattice spacing of 0.219 7 nm. The results of cytotoxicity experiments showed that JYRF-CDs had low toxicity, and the results of animal experiments showed that JYRF-CDs had good anti-inflammatory and analgesic effects. Conclusion: In this study, new type of carbon dots, JYRF-CDs, were discovered after pyrolyzing human hair with motor oil as a dispersant for the first time. Taking JYRF-CDs as a breakthrough, the material base of carbonization products after pyrolysis of human hair by high-temperature pyrolysis using motor oil as a dispersant was more clearly explained, providing a new method for the research of nano compounds.