Water Soluble Propolis and Royal Jelly Enhance the Antimicrobial Activity of Honeys and Promote the Growth of Human Macrophage Cell Line

Due to the overuse and misuse of antibiotic, an increase in antibiotic resistance of pathogenic bacteria is evolving. Attention should be focused on natural alternatives to antibiotics, like propolis, royal jelly （R J） and honeys. They all have strong antibacterial properties due to the active substances they contain. This study investigated the effect of combination of water soluble propolis （WSP） Greitl20 or fresh royal jelly （F-RJ） （MiZigoj） and Forest honeys as antibacterial against Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinetobacter baumanii, Staphylococcus aureus, methicillin resistant Staphylococcus aureus （MRSA）, Streptococcus pyogenes, Streptococcus agalactiae and Candida albicans. These substances are also cell growth promoters for human macrophage （TLT） cell line. WSP Greitl20, F-RJ （M） and different Forest honeys were prepared in saline as 10% solutions. The antimicrobial activity was expressed as the minimal inhibitory concentration （MIC） in mg/mL. The growth promotion activity was measured at optical density （OD） 595 nm. The combination ofWSP Greitl20 with different Forest honeys is better than F-RJ （M） in same combination with different Forest honeys. The best antibacterial/antifungal activity was found with the combination of 10% WSP Greit 120 in the Forest honey （1：10） from Italy or Spain. When measuring the growth promoting activity of TLT cell line, the best activity was detected at the combination of 10% WSP Greitl20 in the Forest honey from Italy （GI3 = 0.796 ± 0.014 and GI5 = 1.133± 0.022）. Antimicrobial and growth promoting activities are correlated and WSP-dependent.

Anti-inflammatory effects of diaporisoindole B in LPS-stimulated RAW 264.7 macrophage cells via MyD88 activated NF-κB and MAPKs pathways

Diaporisoindole B(DPB),an isoprenylisoindole alkaloid isolated from the mangrove endophytic fungus Diaporthe sp.SYSU-HQ3,has been proved to inhibit the production of nitric oxide(NO)in lipopolysaccharide(LPS)-challenged RAW 264.7 mouse macrophages,showing potent anti-inflammatory effects.In this study,we further investigated the anti-inflammatory effects of DPB and explored the possible mechanisms in LPS-challenged RAW 264.7 mouse macrophages.The results showed that DPB(3.125,6.2,12.5 and 25μM)could significantly reduce LPS-induced levels of PGE2,and inhibit the expressions of i NOS and COX-2 in a dose-dependent manner.In addition,DPB also inhibited LPS-induced production of inflammatory cytokines,including TNF-α,IL-1β,IL-6.Moreover,we further investigated signal transduction mechanisms by which DPB exerted anti-inflammatory effects.DPB could affect LPS-mediated nuclear factor kappa B(NF-κB)signaling pathway activation via down-regulating the upstream myeloid differentiation protein 88(MyD88)at the protein level.Additionally,DPB also strongly inhibited the phosphorylation of mitogen-activated protein kinases(MAPKs),including extracellular signal-regulated kinase(ERK)1/2,c-Jun N-terminal kinase(JNK)and p38.Therefore,DPB might exert anti-inflammatory effects by suppressing NF-κB activation and MAPKs pathways via down-regulating MyD88 in RAW 264.7 cells.

Berberine promotes the development of atherosclerosis and foam cell formation by inducing scavenger receptor A expression in macrophage

Berberine is identified to lower the serum cholesterol level in human and hamster through the induction of low density lipoproteins （LDL） receptor in hepatic cells. To evaluate its potential in preventing atherosclerosis, the effect of berberine on atherosclerosis development in apolipoprotcin E-deficient （apoE^-/-） mice was investigated. In apoE^-/- mice, berberine induced in rivo foam cell formation and promoted atheroselerosis development. The foam cell formation induced by berberinc was also observed in mouse RAW264.7 cells, as well as in mouse and human primary macrophages. By inducing scavenger receptor A （SR-A） expression in macrophages, berberine increased the uptake of modified LDL （DiO-Ac-LDL）. Bcrberine-induced SR-A expression was also observed in macrophage foam cells in vivo and in the cells at atherosclerotic lesion. Analysis in RAW264.7 cells indicated that berberine induced SR-A expression by suppressing PTEN expression, which led to sustained Akt activation. Our results suggest that to evaluate the potential of a cholesterol-reducing compound in alleviating atherosclerosis, its effect on the ceils involved in atherosclerosis development, such as macrophages, should also be considered. Promotion of foam cell formation could counter-balance the beneficial effect of lowering serum cholesterol.

MicroRNA sequences modulating inflammation and lipid accumulation in macrophage “foam” cells: Implications for atherosclerosis

Accumulation of macrophage"foam"cells,laden with cholesterol and cholesteryl ester,within the intima of large arteries,is a hallmark of early"fatty streak"lesions which can progress to complex,multicellular atheromatous plaques,involving lipoproteins from the bloodstream and cells of the innate and adaptive immune response.Sterol accumulation triggers induction of genes encoding proteins mediating the atheroprotective cholesterol efflux pathway.Within the arterial intima,however,this mechanism is overwhelmed,leading to distinct changes in macrophage phenotype and inflammatory status.Over the last decade marked gains have been made in understanding of the epigenetic landscape which influence macrophage function,and in particular the importance of small non-coding micro-RNA(miRNA)sequences in this context.This review identifies some of the miRNA sequences which play a key role in regulating"foam"cell formation and atherogenesis,highlighting sequences involved in cholesterol accumulation,those influencing inflammation in sterol-loaded cells,and novel sequences and pathways which may offer new strategies to influence macrophage function within atherosclerotic lesions.

Macrophage polarization in nerve injury： do Schwann cells play a role？

In response to peripheral nerve injury, the inflammatory response is almost entirely comprised of infiltrating macrophages. Macrophages are a highly plastic, heterogenic immune cell, playing an indispensable role in peripheral nerve injury, clearing debris and regulating the microenvironment to allow for efficient regeneration. There are several cells within the microenvironment that likely interact with macrophages to support their function – most notably the Schwann cell, the glial cell of the peripheral nervous system. Schwann cells express several ligands that are known to interact with receptors expressed by macrophages, yet the effects of Schwann cells in regulating macrophage phenotype remains largely unexplored. This review discusses macrophages in peripheral nerve injury and how Schwann cells may regulate their behavior.

Essential Gene(s) Targeted by Peptide Nucleic Acids Kills <i>Mycobacterium smegmatis</i>in Culture and in Infected Macrophages

Background: Antisense peptide nucleic acids (PNAs) exhibit growth inhibitory effects on bacteria by inhibiting the expression of essential genes and could be promising therapeutic agents for treating bacterial infections. A study was carried out to determine the efficacy of several antisense PNAs in inhibiting extracellular and intracellular growth of Mycobacterium smegmatis. Methods: Six PNAs obtained from a commercial supplier were tested to evaluate the inhibitory effect on bacterial growth by inhibiting the expression of the following essential genes: inhA (a fatty acid elongase), rpsL (ribosomal S12 protein), gyrA (DNA gyrase), pncA (pyrazinamidase), polA (DNA polymerase I) and rpoC (RNA polymerase β subunit) of M. smegmatis. Each PNA was tested at 20 μM, 10 μM, 5 μM and 2.5 μM concentrations to determine whether they caused a dose dependent killing of M. smegmatis cultured in Middlebrook 7H9 broth or in a J774A.1 murine macrophage cell line. Results: In Middlebrook broth, the strong growth inhibitory effect against M. smegmatis was observed by PNAs targeting the inhA and rpsL genes at all four concentrations. The PNAs targeting the pncA, polA and rpoC genes were found to exhibit strong growth inhibition against M. smegmatis but only at 20 μM concentration. No growth inhibition of M. smegmatis was seen in pure culture when treated with PNAs targeting gyrA and a mismatch PNA targeting dnaG (DNA primase). All six PNAs showed killing of M. smegmatis in J774A.1 macrophage cell line that were statistically significant (p < 0.05). Conclusion: It may be concluded from this study that PNAs could be potential therapeutics for mycobacterial infections.

Host immune cellular reactions in corneal neovascularization

Corneal neovascularization（CNV） is a global important cause of visual impairment. The immune mechanisms leading to corneal heme- and lymphangiogenesis have been extensively studied over the past years as more attempts were made to develop better prophylactic and therapeutic measures. This article aims to discuss immune cells of particular relevance to CNV, with a focus on macrophages, Th17 cells, dendritic cells and the underlying immunology of common pathologies involving neovascularization of the cornea. Hopefully, a thorough understanding of these topics would propel the efforts to halt the detrimental effects of CNV.

Effects of Sanchi extract on the activation and proliferation of murine lymphocytes and NO secretion by peritoneal macrophages in vitro

The aim of this study is to elucidate the molecular and cellular mechanisms underlying the immunosuppressive effect of Sanchi extract （SE） via investigating the effects of SE on the activation and proliferation of murine lymphocytes and NO secretion by peritoneal macrophages in vitro. ConA was used to activate lymphecytes, and expression of CD69 on T cells and CFSE labeled cell division were detected by flow cytometry. Murine peritoneal macrophages were stimulated with LPS or lymphocytes culture supernate （LCS） and the concentration of NO was determined by Griess reagent assay. After 6 h of culture, SE ranging from 50 to 100μg/ml downregulated CD69 expression on ConA-activated T cells, while SE ranging from 12.5 to 100μg/ml inhibited the proliferative response of lymphocytes to ConA. Additionally, SE （12.5-100μg/ml） inhibited secretion of NO by peritoneal macrophages stimulated by LPS or LCS. This study reveals that SE inhibits the activation and proliferation of routine lymphocytes and NO secretion by peritoneal macrophages.

Neddylation suppression by a macrophage membrane-coated nanoparticle promotes dual immunomodulatory repair of diabetic wounds

Oxidative stress,infection,and vasculopathy caused by hyperglycemia are the main barriers for the rapid repair of foot ulcers in patients with diabetes mellitus(DM).In recent times,the discovery of neddylation,a new type of post-translational modification,has been found to regulate various crucial biological processes including cell metabolism and the cell cycle.Nevertheless,its capacity to control the healing of wounds in diabetic patients remains unknown.This study shows that MLN49224,a compound that inhibits neddylation at low concentrations,enhances the healing of diabetic wounds by inhibiting the polarization of M1 macrophages and reducing the secretion of inflammatory factors.Moreover,it concurrently stimulates the growth,movement,and formation of blood vessel endothelial cells,leading to expedited healing of wounds in individuals with diabetes.The drug is loaded into biomimetic macrophage-membrane-coated PLGA nanoparticles(M-NPs/MLN4924).The membrane of macrophages shields nanoparticles from being eliminated in the reticuloendothelial system and counteracts the proinflammatory cytokines to alleviate inflammation in the surrounding area.The extended discharge of MLN4924 from M-NPs/MLN4924 stimulates the growth of endothelial cells and the formation of tubes,along with the polarization of macrophages towards the anti-inflammatory M2 phenotype.By loading M-NPs/MLN4924 into a hydrogel,the final formulation is able to meaningfully repair a diabetic wound,suggesting that M-NPs/MLN4924 is a promising engineered nanoplatform for tissue engineering.

Effects of Andrographolide on the Activation of Mitogen Activated Protein Kinases and Nuclear Factor-κ B in Mouse Peritoneal Macrophage-derived Foam Cells

Objective： To observe the effect of andrographolide on the activation of mitogen-activated protein kinases （MAPKs） and expression of nuclear factor- kB （NF-kB） in macrophage foam cells. Methods： The mouse peritoneal macrophages were cultured in the media in the presence of oxidized low-density lipoprotein （ox-LDL）, ox-LDL＋andrographolide, or neither （control）. The phosphorylation of MAPK molecules （p38MAPK, JNK, ERK1/2） and the expressions of NK- kB p65 were examined by Western blot. Results： As compared with cells in the control group, the expressions of phospho-p38 and NF- kB p65 were increased in the cells cultured with either ox-LDL or ox-LDL＋andrographolide （P〈0.01）, but attenuated significantly in the presence of ox-LDL＋ andrographolide when compared with ox-LDL （P〈0.05）. The phospho-JNK increased in the presence of either ox-LDL or ox-LDL＋andrographolide when compared with control cells （P〈0.01）, but no significant difference existed between ox-LDL and ox-LDL＋andrographolide （P〉0.05）. The expression of phospho-ERK1/2 was increased in the presence of ox-LDL compared with the control cells （P〈0.01）, but no significant differences existed between the cells cultured in the presence of ox-LDL＋andrographolide and the control medium （P〉0.05）. Conclusions： Andrographolide could inhibit the activation of ERK1/2, p38MAPK and NK-kB induced by ox-LDL in macrophage foam cells, which might be one of its mechanisms in preventing atherosclerosis.

Time-dependent gene expression analysis after mouse skeletal muscle contusion

Background:Though the mechanisms of skeletal muscle regeneration are deeply understood,those involved in muscle contusion,one of the most common muscle injuries in sports medicine clinics,are not.The objective of this study is to explore the mechanisms involved in muscle regeneration after contusion injury.Methods:In this study,a total of 72 mice were used.Eight of them were randomly chosen for the control group,while the rest were subjected to muscle contusion.Subsequently,their gastrocnemius muscles were harvested at different time points.The changes in muscle morphology were assessed by hematoxylin and eosin(HE) stain.In addition,the gene expression was analyzed by real-time polymerase chain reaction.Results:The data showed that the expression of many genes,i.e.,specific markers of immune cells and satellite cells,regulatory factors for muscle regeneration,cytokines,and chemokines,increased in the early stages of recovery,especially in the first 3 days.Furthermore,there were strict rules in the expression of these genes.However,almost all the genes returned to normal at 14 days post-injury.Conclusion:The sequence of immune cells invaded after muscle contusion was neutrophils,M1 macrophages and M2 macrophages.Some CC(CCL2,CCL3,and CCL4) and CXC(CXCL10) chemokines may be involved in the chemotaxis of these immune cells.HGF may be the primary factor to activate the satellite cells after muscle contusion.Moreover,2 weeks are needed to recover when acute contusion happens as used in this study.

Immunomodulation of polypeptides fromChlamys farreri in vitro

The aim of this study was to investigate the effects of polypeptides from Chlamys farreri （PCF） on immunoeytes or immunocytes treated with dexamethasone （DEX） in vitro. After incubating immunoeytes with 25 mg/L PCF or/and DEX for a given time, the proliferative response of thymocytes and splenoeytes to ConA were measured bv MTF assay; the subpopulations of thymocytes and splenic T lymphoeytes was analyzed by flow cytomety; the cytotoxicity of natural killer cells was measured by Llactate dehydrogenase （LDH） assay; the phagocytosis of rat peritoneal macrophages was measured by Neutral red assay and the Bel-2 protein expression of macrophages was detected by imrnunocytoehemical stain. The proliferative ability of rat thymocytes and splenocytes induced with ConA was enhanced and the depression of lymphoproliferation caused by DEX was reversed by PCF. The percentages of mouse thymic L3 T4^- Lyt-2^- and Lyt-2^＋ subpopulations and splenic Lyt-2 ^＋ cells were decreased and the percentage of splenic L3 T4^ ＋ cells was increased by PCF. The NK cytotoxicity, phagocytosis of macraphages and Bcl-2 protein expression of macrophages were enhanced and the decrease of NK cytotoxicity and Bel-2 protein expression of maerophages caused by DEX were reversed by PCF. PCF could not only enhance the normal immunity function, but also reverse the imrnunosuppression induced by DEX.

Pentoxifylline Inhibits Liver Fibrosis via Hedgehog Signaling Pathway

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog（HH） signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline（PTX） on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate（PMA） was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen（SEA）, and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8（CCK-8） was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and（or） PTX to detect the transforming growth factor-β gene expression. The m RNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The m RNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells（HSCs）. The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.

A pH-responsive biomimetic drug delivery nanosystem for targeted chemo-photothermal therapy of tumors

Smart drug delivery nanosystem is significant for tumor treatments due to its possibility of temporally,spatially,and dose-controlled release.However,the therapeutic efficacy of drug delivery nanosystem is often compromised in cancer treatment as the enrichment of therapeutic agents in the reticuloendothelial system.Herein,doxorubicin(DOX)loaded biomimetic drug delivery nanosystem with macrophage cell membrane(MCM)camouflaged,MnFe_(2)O_(4)-DOX-MCM nanocube(NC),is developed for cancer treatment with tumor targeting,pH-stimuli drug release,and chemo-photothermal therapeutic effects.The nanosystem shows the capability of immune escape and enhanced cellular uptake of cancer cells due to the MCM decoration.Acid-labile bond between the MnFe2O4 NCs and DOX remains stable at physiological condition and release drugs immediately in response to the endo-/lysosome pH stimuli.Meanwhile,the photothermal effect of the nanosystem destroys tumor tissue,which further promotes chemotherapeutic efficacy.In vivo results demonstrate the tumor homing ability and produce a notable synergistic therapeutic effect of the NCs.Thus,biomimetic pH-responsive drug delivery nanosystem,MnFe_(2)O_(4)-DOX-MCM NCs,is an effective nanoplatform,which might be potential application for cancer synergistic treatment.

Inflammatory Changes in Paravertebral Sympathetic Ganglia in Two Rat Pain Models

Injury to peripheral nerves can lead to neuropathic pain, along with well-studied effects on sensory neurons, including hyperexcitability, abnormal spontaneous activity, and neuroinflammation in the sensory ganglia. Neuropathic pain can be enhanced by sympathetic activity. Peripheral nerve injury may also damage sympathetic axons or expose them to an inflammatory environment. In this study, we examined the lumbar sympathetic ganglion responses to two rat pain models： ligation of the L5 spinal nerve, and local inflammation of the L5 dorsal root ganglion （DRG）, which does not involve axotomy. Both models resulted in neuroinflammatory changes in the sympathetic ganglia, as indicated by macrophage responses, satellite glia activation, and increased numbers of T cells, along with very modest increases in sympathetic neuron excitability （but not spontaneous activity） measured in ex vivo recordings. The spinal nerve ligation model generally caused larger responses than DRG inflammation. Plasticity of the sympathetic system should be recognized in studies of sympathetic effects on pain.

Resolution of inflammation and repair after ischemic brain injury

After ischemic stroke, proinflammatory molecules known as danger-associated molecular patterns (DAMPs) originating from damaged brain cells recruit and activate immune cells (neutrophils, macrophages, lymphocytes) further eliciting innate and adaptive immunity. During the acute phase from day 1 to day 3 of the stroke onset, macrophages play a major role in the progression of inflammation, promoting the destruction of brain tissue. During the recovery phase, from day 3~4 to day 7 after stroke onset, infiltrating macrophages switch to repairing macrophages, which clear the DAMPs and promote tissue repair by producing neurotrophic factors. Adaptive immunity during the late or chronic phase (> day 7) of stroke has not been well investigated. Recent studies have also indicated that antigen-specific T cells, especially regulatory T cells (Tregs), play major roles in neural repair. This review focuses mainly on the resolution of inflammation and tissue repair by macrophages and Tregs.