Xiaojindan Extract Modulated Macrophage Polarization by Targeting PI3K/Akt Pathway / 中国实验方剂学杂志

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.

MFN1 ubiquitination mediates lipopolysaccharide-induced mitochondrial dysfunction and pyroptosis in Raw264.7 mouse macrophages / 中华微生物学和免疫学杂志

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.

Interleukin-1 alpha induces osteoclast activation and bone loss / 中国组织工程研究

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.

A new benzaldehyde from the coral-derived fungus Aspergillus terreus C23-3 and its anti-inflammatory effects via suppression of MAPK signaling pathway in RAW264.7 cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Screening of Anti-inflammatory Active Sites of Water Extract of Iris halophila Root and Analysis of Its Chemical Constituents / 中国实验方剂学杂志

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.

Inhibitory Effect and Mechanism of Jingulian Extract on LPS-induced RAW264.7 Cell Inflammatory Response Based on PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

Objective:To explore the inhibitory effect and mechanism of Jingulian extract （JGL） on inflammation. Method:The following groups were set up in this study： a control group （10% fetal bovine serum）， a lipopolysaccharide （LPS） model group （0.5 mg·L^-1）， and JGL groups （10， 20， 40， 60， 80， 120， 160， 200， 250， 300 mg·L^-1 + 0.5 mg·L^-1LPS）. The RAW264.7 cells were cultured for 24 hours. Cell proliferation was detected by cell counting kit-8 （CCK-8） assay. Nitric oxide （NO） release was detected by Griess assay. The release of cytokines interleukin （IL）-1<italic>β</italic>， IL-6， IL-10， and tumor necrosis factor （TNF）-<italic>α</italic> was determined by enzyme linked immunosorbent assay （ELISA）. The expression of inducible nitric oxide synthase （iNOS） and intraprostaglandin peroxidase synthase 2 （PTGS2）/cyclooxygenase-2 （COX-2） was measured by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） and the activation of key proteins in the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway by Western blot. Result:Compared with the control group， LPS （0.5 mg·L^-1）could promote the proliferation of RAW264.7 cells after stimulation for 24 hours （<italic>P</italic><0.01）. Compared with the model group， JGL had no significant effect on cell proliferation. Compared with the control group， LPS （0.5 mg·L^-1）increased the release of NO， IL-1<italic>β</italic>， IL-6， IL-10， and TNF-<italic>α</italic> （<italic>P</italic><0.01）. Compared with the model group， JGL （20-300 mg·L^-1）inhibited the release of NO in a dose-dependent manner after stimulation for 24 hours （<italic>P</italic><0.05） and reduced IL-1<italic>β</italic>， IL-6， and IL-10 （<italic>P</italic><0.05， <italic>P</italic><0.01）， but no obvious inhibition on the release of TNF-<italic>α</italic> was observed. LPS （0.5 mg·L^-1） could induce the expression of iNOS and PTGS2/COX-2 genes as compared with the control group （<italic>P</italic><0.05， <italic>P</italic><0.01）. JGL could down-regulate the mRNA expression of iNOS and PTGS2/COX-2 genes as compared with the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）. LPS （0.5 mg·L^-1） could activate the PI3K/Akt pathway （<italic>P</italic><0.01） as compared with the control group， while JGL （10， 20， 40， and 80 mg·L^-1） decreased the expression of PI3K-p110， p-p85， and p-Akt （<italic>P</italic><0.01）， and inhibited the activation of PI3K/Akt pathway. Conclusion:JGL extract could significantly inhibit the inflammatory response and activation of the PI3K/Akt pathway induced by LPS in RAW264.7 cells. The anti-inflammatory effect was related to the inhibition of the PI3K/Akt pathway.

Biological Evaluation for Quality Control of Water Extract of Qingjin Huatantang Based on Phagocytic and Secretory Functions of Macrophages / 中国实验方剂学杂志

Objective:To establish a method for evaluating the biological activity of water extract lyophilized powder of Qingjin Huatantang based on the phagocytic and secretory functions of macrophages， and to control the quality of this formula from the biological activity level. Method:The phagocytic and inflammation models of RAW264.7 macrophages were established， the inhibition rates of water extract lyophilized powder of Qingjin Huatantang on interleukin-6 （IL-6） secretion and phagocytic index of neutral red of RAW264.7 macrophages were chosen as indicators to investigate the biological activity of Qingjin Huatantang， and the biological limit was searched. Result:The optimal inoculation density of RAW264.7 macrophages was 3×10⁵ pcs/mL， and the concentration of lipopolysaccharide （LPS） was 1 mg·L^-1 after treatment for 24 h. When the concentration was 500 mg·L^-1， water extract lyophilized powder of Qingjin Huatantang had no toxicity and no obvious promotion effect on the proliferation of RAW264.7 macrophages， and at this concentration， the phagocytosis of RAW264.7 macrophages for neutral red was significantly promoted， the phagocytic index was >113%. In addition， the lyophilized powder had a significant and stable inhibitory effect on IL-6 secretion of RAW264.7 macrophages induced by LPS， the inhibitory rate was >45%. Conclusion:Combined with the anti-inflammatory and immunomodulatory effects of Qingjin Huatantang， this study establishes an <italic>in vitro </italic>biological limit method for evaluating the quality of water extract of Qingjin Huatantang based on the phagocytic and secretory functions of RAW264.7 macrophages， and 500 mg·L^-1 was confirmed as the limit concentration. Under the limit concentration， Qingjin Huatantang water extract can significantly promote the phagocytic index of macrophages or significantly inhibit the secretion of IL-6 of RAW264.7 macrophages induced by LPS， which can be judged as qualified.

Immunoregulatory effect of polysaccharides derived from chroogomphus rutilus on macrophage cell line RAW264.7 / 药学实践杂志

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.

Protective Effect of Brassica napus L. Hydrosols against Inflammation Response in RAW 264.7 Cells / 中国结合医学杂志

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.

In Vitro Anti-Inflammatory And Hepatoprotective Activity Of Turmesac®

Objective: In this study, we investigated the hepatoprotective activity of Turmesac® on Human liver cells (HepG2 cell line) and anti-inflammatory effect on Murine macrophages (Raw 264.7 cell line) by flow Cytometry. Methods: Cell viability of HepG2 and Raw 264.7 cells determined by the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay to identify a non-cytotoxic concentration of Turmesac® for the respective cell lines after 24 h exposure period. Further hepatoprotective effect of Turmesac® was performed in H2O2 treated liver cells using H2DCF-DA staining by flow cytometry. The anti-inflammatory potency of Turmesac® was evaluated in Lipopolysaccharide (LPS 2µg/ml) stimulated Murine Raw 264.7 macrophages by measuring the relative fluorescence intensity of 2 cytokines, Interleukin-8(IL-8) and (Interleukin-12) IL-12 by flow cytometric analysis. Results: Turmesac® concentrations of less than 50μg/ml did not show significant cytotoxicity on both HepG2 and Raw 264.7, cell lines following the treatment period of 24 h and selected 50μg/ml as the optimum concentration for hepatoprotective and anti-inflammatory models. The reactive oxygen species (ROS) study revealed that Turmesac® (50μg/ml) effectively suppressed the H2DCF-DA expression in HepG2 cells. Secondly, Turmesac® significantly suppressed the anti-inflammatory cytokine expressions of IL-8 and IL-12 in LPS pre-stimulated cells categorising as a potentially potent anti-inflammatory drug. The mean fluorescence intensity percentage of IL-8 is control 8.86, LPS 50.49, Turmesac® 19.63 and IL12 is control 10.41, LPS 68.94, and Turmesac® 15.79 respectively. Conclusion: This study highlighted that Turmesac® could be considered as a promising hepatoprotective and anti-inflammatory compound and a therapeutic agent in curing liver-related and inflammation-related diseases.

Antioxidant and anti-inflammatory activities of Orostachys japonicus

Objective: To determine the bioactive compounds of Orostachys japonicus (O. japonicus) (Maxim.) A. Berger extraction by different solvents (70％ ethanol or water) and to evaluate the in vitro antioxidant and anti-inflammatory activities.Methods: Total polyphenol, flavonoid, and anthocyanin contents in O. japonicus extract were measured. Antioxidant activities were determined by DPPH, ABTS, and superoxide radical-scavenging ability assays. Anti-inflammatory activities were evaluated by nitric oxide production, tumor necrosis factor-α, and interleukin-6 expression techniques. Results: Extraction with 70％ ethanol yielded the highest total polyphenol (60.03 mg/g dry weight) and flavonoid (55.50 mg/g dry weight) contents. The total anthocyanin contents in 70％ ethanol and water extracts were 57.25 and 91.71 mg/g dry weight, respectively. The 70％ ethanol extract also showed stronger antioxidant activity than the water extract. Antioxidant activity and reducing power increased with the increasing concentration of O. japonicus extract. O. japonicus extract at 0-400 μg/mL did not affect the growth of RAW 264.7 cells, whereas dose-dependent inhibition of nitric oxide, tumor necrosis factor-α, and interleukin-6 production was observed. Conclusions: O. japonicus inhibits oxidative and inflammatory reactions with potentially positive health-related effects.

Intervention Effect of Shenfu Injection on the Nuclear Translocation of HMGB 1 in LPS-induced RAW 264.7 Cells / 中国药房

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.

Caulerpa okamurae extract attenuates inflammatory interaction, regulates glucose metabolism and increases insulin sensitivity in 3T3-L1 adipocytes and RAW 264.7 macrophages / 中西医结合学报

OBJECTIVE@#To examine whether Caulerpa okamurae ethanolic extract (COE) could inhibit obesity-mediated inflammation, improve glucose metabolism and increase insulin sensitivity, using in vitro cell models of RAW 264.7 macrophages and 3T3-L1 adipocytes.@*METHODS@#We cocultured 3T3-L1 adipocytes in direct contact with lipopolysaccharide-stimulated RAW 264.7 macrophages and induced insulin resistance in 3T3-L1 adipocytes with tumor necrosis factor-α (TNF-α) in the presence or absence of 250 µg/mL of COE. We investigated various markers of inflammation, glucose regulation and insulin sensitivity in these models using Griess reagent to measure nitric oxide (NO) production, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose to measure glucose uptake, Western blot analysis to quantify protein expression and reverse transcriptase-polymerase chain reaction to evaluate mRNA expression.@*RESULTS@#We found that COE (250 µg/mL) significantly inhibited the lipopolysaccharide-induced inflammatory response in RAW 264.7 macrophages by downregulating NO production, nitric oxide synthase 2 expression and nuclear translocation of nuclear factor-κB. COE also showed similar anti-inflammatory activity in coculture, along with decreased TNF-α, interleukin-6 and monocyte chemoattractant protein mRNA expression. In addition, COE also improved glucose uptake in coculture by upregulating glucose transporter-4 (GLUT-4) and adiponectin and reducing serine phosphorylation of insulin receptor substrate-1 (IRS1). In the TNF-α-induced insulin resistance model of 3T3-L1 adipocytes, COE significantly improved both basal and insulin-stimulated glucose uptake, accompanied by phosphorylation of IRS1 at tyrosine 632, phospho-5' adenosine monophosphate-activated protein kinase α and glycogen synthase kinase-3β (Ser9) as well as upregulation of GLUT-4.@*CONCLUSION@#Together, these findings suggest that COE has potential to treat or prevent obesity-induced metabolic disorders.

Microrna-21 regulates proliferation and differentiation of osteoclasts in periodontitis / 中国组织工程研究

BACKGROUND: MicroRNA-21 (miR-21) is a regulator of osteoclastogenesis and a promoter of osteoclast differentiation, but its role in periodontitis remains unclear. OBJECTIVE: To investigate whether miR-21 is involved in bone destruction in periodontitis. METHODS: Real-time PCR was used to detect and analyze the differential expression of miR-21 in periodontitis samples. Using liposome transfection method, miR-21 mimics (up-regulating miR-21) or miR-21 inhibitor (down-regulating miR-21) was used to transfect osteoclasts. Expressions of miR-21 and bone destruction markers TRAP and CTSK were detected by real-time PCR. Cell counting kit-8 was used to detect the miR-21 effect on osteoclast proliferation. RESULTS AND CONCLUSION: (1) MiR-21 expression increased in periodontitis samples. (2) When miR-21 mimics was transfected into osteoclasts, miR-21, TRAP and CTSK mRNA expression increased; when miR-21 inhibitor was transfected into osteoclasts, miR-21, TRAP and CTSK mRNA expression decreased. (3) Transfection with miR-21 mimics promoted the proliferation of osteoclasts, while transfection with miR-21 inhibitor inhibited the proliferation of osteoclasts. To conclude, miR-21 can be used as an important target for the treatment of periodontitis.

Inhibition of bakuchiol and its derivatives in Psoraleae Fructus on nitric oxide production in lipopolysaccharide-activated RAW 264.7 cell lines / 中草药

Objective: To study bakuchiol and its derivatives of cyclohexane soluble part in 70% ethanol aqueous extract of Psoraleae Fructus and their inhibition on nitric oxide (NO) production in lipopolysaccharides (LPS)-activated murine macrophage RAW 264.7 cell lines. Methods: The compounds were separated and purified by silica gel column and high performance liquid chromatographies, and their structures were determined by spectroscopic data analyses. Using LPS-activated RAW 264.7 cell line models in vitro, all of the isolated compounds were evaluated for the inhibition against NO production. Results: Twelve compounds were obtained and identified as bakuchiol (1), 12,13-dihydro-12,13-epoxybakuchiol (2), Δ3,2-hydroxylbakuchiol (3), 12-oxobakuchiol (4), psoracorylifol B (5), psoracorylifol C (6), (12’S)-bisbakuchiol C (7), Δ1,3-bakuchiol (8), 13-methoxyisobakuchiol (9), bisbakuchiol B (10), bisbakuchiol A (11), and 12,13-dihydro-12,13-dihydroxybakuchiol (12), respectively. For the inhibition of NO production in the LPS-activated RAW 264.7 cell line model, a positive inhibitor, L-N6-(1-iminoethyl)-lysine (L-NIL), was used and showed the half maximal inhibitory concentration (IC50) value of (10.29 ± 1.10) μmol/L. The IC50 values of the assayed compounds 1, 3, 5, 10 and 11 were all more than 50 μmol/L, compounds 8, 9 and 12 were comparable to that of L-NIL, whereas the IC50 values of compounds 2, 4 and 7 were less than that of the positive inhibitor with statistically significance. Conclusion: Compound 4 is a new natural product. The results of the bioactivity assays indicated that compounds 2, 4, 7, 8, 9 and 12 are potential anti-inflammatory agents.

Synthesis of a new type of carbon dots by pyrolysis of human hair using motor oil as a dispersant and their biological effects / 中草药

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.

Effects of advanced glycation end products on osteoclasts at different stages of differentiation / 南方医科大学学报

OBJECTIVE@#To explore the effect of advanced glycation end products (AGEs) on osteoclasts at different stages of differentiation.@*METHODS@#Raw264.7 cells cultured were induced for osteoclastogenesis using RANKL, and the stages of differentiation of the osteoclasts were determined with TRAP staining. The cells were then randomly divided into control group, early-stage AGEs intervention group and late-stage AGEs intervention group. The viability of the cells after AGEs treatment was assessed using CCK-8 method. The cells were examined after the induction for osteoclastogenesis using TRAP staining, and the expression levels of RANK, NFATC-1, TRAF-6, TRAP and CTSK mRNAs were tested with RT-PCR; the expressions of CTSK and RANK proteins were detected using Western boltting.@*RESULTS@#We defined the initial 3 days of induction as the early stage of differentiation and the time beyond 3 days as the late stage of differentiation of Raw264.7 cells. Intervention with AGEs at 100 mg/L produced no significant effects on the viability of the cells, but AGEs suppressed the cell proliferation at a concentration exceeding 100 mg/L. The number of osteolasts in the early- and late-stage intervention groups was greater than that in the control group, but the cell count differed significantly only between the early-stage intervention group and control group ( < 0.05). The gene expressions of RANK, NFATC-1, TRAF-6, TRAP and CTSK all increased after the application of AGEs in both the early and late stages of differentiation, but the changes were significant only in the early-stage intervention group ( < 0.05). The changes in CTSK and RANK protein expressions were consistent with their mRNA expressions.@*CONCLUSIONS@#AGEs can affect the differentiation of osteoclasts differently when applied at different stages, and intervention with AGEs at the early stage produces stronger effect to promote osteoclast differentiation than its application at a late stage.

Study on anti-inflammatory active components and mechanism of Corydalis Bungeanae Herba / 中国中药杂志

Corydalis Bungeanae Herba is often used to treat a variety of inflammatory diseases in traditional Chinese medicine. In order to determine its chemical material basis, the components of Corydalis Bungeanae Herba were isolated by automated purification system. Flavonoids and alkaloids were prepared, and all such components were identified by mass spectrometry. The effects of the components on the production of inflammatory mediators and pharmacological mechanisms in the lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model were examined. Mouse macrophages(RAW264.7) were first treated with LPS. The relationship between cell viability and LPS concentration was observed. Then, the effects of flavonoids components and alkaloid components with different administration concentrations on cell viability were detected to determine the maximum administration concentration. Secondly, 2.5, 5, 10 and 20 μg·mL~(-1) flavonoids components and alkaloid components were added respectively to observe the effects and mechanism of different concentrations of flavonoids components and alkaloid components on LPS-induced inflammation of RAW264.7 macrophages. Griess reagent assay was used to detect NO content in cell supernatant. The inflammatory cytokines(TNF-α, IL-1β and IL-6) in cell supernatant were determined by ELISA method. Western blot method was used to detect the intracellular nuclear factor(NF-κB) IκBα phosphorylation(p-IκBα), p65 phosphorylation(p-p65) and protein expression of TLR4, TLR2. The results showed that the alkaloid components inhibited the production of NO, TNF-α, IL-1β and IL-6 in a dose-dependent mannerin the concentration range of 2.5-20 μg·mL~(-1). In inflammation upstream pathways, the inhibitory effect of the alkaloid components on the TLR2 expression level was weaker than that of TLR4. In inflammation downstream, alkaloid components significantly inhibited phosphorylation of IκBα and p65 in a dose-dependent manner. These data suggested that the alkaloid components were the material basis components of Corydalis Bungeanae Herba, and its anti-inflammatory mechanism might be related to inhibiting the transmission of inflammatory signals in TLRs/NF-κB signaling pathways dominated by TLR4, interfering with the activation of inflammatory genes and inhibiting their over expression, and down-regulating the secretion level of inflammatory factors.

Inhibitory Mechanisms of Water Extract of Oplopanax elatus on Lipopolysaccharide-Induced Inflammatory Responses in RAW 264.7 Murine Macrophage Cells / 中国结合医学杂志

OBJECTIVE@#To study the anti-inflammatory action and cellular mechanism of Oplopanax elatus.@*METHODS@#A hot water extract of OE (WOE) was prepared and a major constituent, syringin, was successfully isolated. Its content in WOE was found to be 214.0 µg/g dried plant (w/w). Their anti-inflammatory activities were examined using RAW 264.7 macrophages and a mouse model of croton oil-induced ear edema.@*RESULTS@#In lipopolysaccharide (LPS)-treated RAW 264.7 cells, a mouse macrophage cell line, WOE was found to significantly and strongly inhibit cyclooxygenase-2 (COX-2)-induced prostaglandin E (PGE) production [half maximal inhibitory concentration (IC)=135.2 µg/mL] and inducible nitric oxide synthase (iNOS)-induced NO production (IC=242.9 µg/mL). In the same condition, WOE was revealed to inhibit NO production by down-regulating iNOS expression, mainly by interrupting mitogen activated protein kinases (MAPKs)/activator protein-1 (AP-1) pathway. The activation of all three major MAPKs, p38 MAPK, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase, was inhibited by WOE (50-300 µg/mL). On the other hand, WOE reduced PGE production by inhibiting COX-2 enzyme activity, but did not affect COX-2 expression levels. In addition, WOE inhibited the production of proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-α. In croton oil-induced ear edema in mice, oral administration of WOE (50-300 mg/kg) dose-dependently inhibited edematic inflammation.@*CONCLUSION@#Water extract of OE exhibited multiple anti-inflammatory action mechanisms and may have potential for treating inflammatory disorders.

Intervention Effects of Berberine on Mice Macrophage Polarization Based on TLR 4/MyD88/NF-κB Signaling Pathway / 中国药房

OBJECTIVE：To study the effects of berberine on mic e macrophage polarization based on TLR 4-MyD88-NF-κB signaling pathway. METHODS ：Using mice RAW 264.7 macrophage as the object ，atorvastatin calcium as positive control ， inflammatory cell model was induced by lipopolysaccharide （LPS）；ELISA method was used to detect the contents of TNF-α，IL-6 and NF-κB in cell culture medium after treated with low，medium and high doses of berberine （5，10，20 μmol/L）for 24 h. The real-time fluorescence quantitative PCR was conducted to determine the mRNA expression of TLR 4 and MyD 88 in cells. Western blotting assay was used to detect the protein expression of TLR 4，MyD88，iNOS and CD 206 in cells. RESULTS ：Compared with blank control group ，the contents of TNF-α，IL-6 and NF-κB in cell culture medium，mRNA expression of TLR 4 and MyD 88， protein expression of TLR 4，MyD88 and iNOS in cells were increased significantly in LPS induction group （P＜0.05）. Compared with LPS induction group ，the contents of TNF-α and IL-6，mRNA and protein expression of TLR 4 and MyD 88 in atorvastatin calcium group ，berberine medium-dose and high-dose groupsas well as the content of NF-κ B and protein expression of iNOS in administration groups were decreased significantly ， while the content of NF-κB in berberine high-dose group was significantly lower than atorvastatin calcium group （P＜0.05）. The protein expressions of CD206 in atorvastatin calcium group and berberine high-dose group were increased significantly ，while the protein expression of CD 206 in berberine high-dose group was significantly higher than atorvastatin calcium group （P＜0.05）. CONCLUSIONS ：Different doses of berberine can intervene in mice macrophage polarization to different extents ，the mechanism of which may be associated with the regulation of TLR4/MyD88/NF-κB signaling pathway.