Poly(ADP-ribose)polymerase family member 14 promotes functional recovery after spinal cord injury through regulating microglia M1/M2 polarization via STAT1/6 pathway

Poly(ADP-ribose)polymerase family member 14(PARP14),which is an intracellular mono(ADP-ribosyl)transferase,has been reported to promote post-stroke functional recovery,but its role in spinal cord injury(SCI)remains unclear.To investigate this,a T10 spinal cord contusion model was established in C57BL/6 mice,and immediately after the injury PARP14 shRNA-carrying lentivirus was injected 1 mm from the injury site to silence PARP14 expression.We found that PARP14 was up-regulated in the injured spinal cord and that lentivirus-mediated downregulation of PARP14 aggravated functional impairment after injury,accompanied by obvious neuronal apoptosis,severe neuroinflammation,and slight bone loss.Furthermore,PARP14 levels were elevated in microglia after SCI,PARP14 knockdown activated microglia in the spinal cord and promoted a shift from M2-polarized microglia(anti-inflammatory phenotype)to M1-polarized microglia(pro-inflammatory phenotype)that may have been mediated by the signal transducers and activators of transcription(STAT)1/6 pathway.Next,microglia M1 and M2 polarization were induced in vitro using lipopolysaccharide/interferon-γand interleukin-4,respectively.The results showed that PARP14 knockdown promoted microglia M1 polarization,accompanied by activation of the STAT1 pathway.In addition,PARP14 overexpression made microglia more prone to M2 polarization and further activated the STAT6 pathway.In conclusion,these findings suggest that PARP14 may improve functional recovery after SCI by regulating the phenotypic transformation of microglia via the STAT1/6 pathway.

Effect of Pingchuan granule on typeⅡinnate lymphocytes and M2 polarization of macrophages in asthmatic mice

Objective:To observe the effect of Pingchuan granule on the number of typeⅡinnate lymphocytes(ILC2)and M2 polarization of macrophages in the lung tissue of asthmatic mice;Methods:Ovalbumin sensitized and challenged asthmatic mouse models were established,and then Pingchuan granules or IL-33 neutralizing antibody were given to intervene.The pathological morphology of lung tissue was observed by HE,PAS and Masson staining,and the expressions of IL-4,IL-5,IL-13 and IL-33 in BALF and lung tissue were detected by ELISA and qRT-PCR,Flow cytometry was used to detect the number of type II innate lymphocytes and type M2 macrophages in lung tissue.Western blot was used to detect the protein expression levels of ST-2,FIZZ1 and Arg-1 in lung tissue;Results:Compared with the control group,the inflammation score,PAS score and collagen staining area of the model group were significantly increased,the expressions of IL-4,IL-5,IL-13 and IL-33 in BALF and lung tissue were significantly increased,the number of ILC2 and M2 macrophages,the expression of ST-2,FIZZ1 and Arg-1 protein in lung tissue were significantly increased,and the differences were statistically significant(P<0.05);Compared with the model group,Pingchuan granule could significantly reduce the inflammation score,PAS score and collagen staining area of asthmatic mice,down-regulate the expression of IL-4,IL-5,IL-13 and IL-33 in BALF and lung tissue,reduce the number of ILC2 and M2 macrophages,and the expression of ST-2,FIZZ1 and Arg-1 protein in lung tissue(P<0.05);Conclusion:Pingchuan granule improve the airway remodeling of asthma by inhibiting the polarization of M2 macrophages mediated by ILC2.

Calculus bovis inhibits M2 tumor-associated macrophage polarization via Wnt/β-catenin pathway modulation to suppress liver cancer

BACKGROUND Calculus bovis(CB),used in traditional Chinese medicine,exhibits anti-tumor effects in various cancer models.It also constitutes an integral component of a compound formulation known as Pien Tze Huang,which is indicated for the treatment of liver cancer.However,its impact on the liver cancer tumor microenvironment,particularly on tumor-associated macrophages(TAMs),is not well understood.AIM To elucidate the anti-liver cancer effect of CB by inhibiting M2-TAM polarization via Wnt/β-catenin pathway modulation.METHODS This study identified the active components of CB using UPLC-Q-TOF-MS,evaluated its anti-neoplastic effects in a nude mouse model,and elucidated the underlying mechanisms via network pharmacology,transcriptomics,and molecular docking.In vitro assays were used to investigate the effects of CB-containing serum on HepG2 cells and M2-TAMs,and Wnt pathway modulation was validated by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis.RESULTS This study identified 22 active components in CB,11 of which were detected in the bloodstream.Preclinical investigations have demonstrated the ability of CB to effectively inhibit liver tumor growth.An integrated approach employing network pharmacology,transcriptomics,and molecular docking implicated the Wnt signaling pathway as a target of the antineoplastic activity of CB by suppressing M2-TAM polarization.In vitro and in vivo experiments further confirmed that CB significantly hinders M2-TAM polarization and suppresses Wnt/β-catenin pathway activation.The inhibitory effect of CB on M2-TAMs was reversed when treated with the Wnt agonist SKL2001,confirming its pathway specificity.CONCLUSION This study demonstrated that CB mediates inhibition of M2-TAM polarization through the Wnt/β-catenin pathway,contributing to the suppression of liver cancer growth.

Unveiling the anticancer effect of traditional Chinese herbal medicine

In this issue of World Journal of Gastroenterology,Huang et al reported that Calculus bovis(CB),a traditional Chinese herbal medicine,impedes the growth of liver cancers in vivo.Through further in vitro studies,they showed that CB suppressed the M2 polarization of tumor-associated macrophages by suppressing the Wnt signaling pathway,which consequently inhibited the growth of liver cancer.Although the effects of traditional Chinese herbal medicine are often not scientifically proven,Huang et al successfully identified the molecular mechanism involved in the anticancer effect of CB,and it is anticipated that the molecular mechanisms involved in the effects of other traditional Chinese herbal medicines will be scientifically elucidated,as demonstrated in this article.

Exosomal microRNA-588 from M2 polarized macrophages contributes to cisplatin resistance of gastric cancer cells

BACKGROUND Gastric cancer is a prevalent malignant cancer with a high incidence and significantly affects the health of modern people globally.Cisplatin(DDP)is one of the most common and effective chemotherapies for patients with gastric cancer,but DDP resistance remains a severe clinical challenge.AIM To explore the function of M2 polarized macrophages-derived exosomal microRNA(miR)-588 in the modulation of DDP resistance of gastric cancer cells.METHODS M2 polarized macrophages were isolated and identified by specific markers using flow cytometry analysis.The exosomes from M2 macrophages were identified by transmission electron microscopy and related markers.The uptake of the PKH67-labelled M2 macrophages-derived exosomes was detected in SGC7901 cells.The function and mechanism of exosomal miR-588 from M2 macrophages in the modulation of DDP resistance of gastric cancer cells was analyzed by CCK-8 assay,apoptosis analysis,colony formation assay,Western blot analysis,qPCR analysis,and luciferase reporter assay in SGC7901 and SGC7901/DDP cells,and by tumorigenicity analysis in nude mice.RESULTS M2 polarized macrophages were isolated from mouse bone marrow stimulated with interleukin(IL)-13 and IL-4.Co-cultivation of gastric cancer cells with M2 polarized macrophages promoted DDP resistance.M2 polarized macrophagesderived exosomes could transfer in gastric cancer cells to enhance DDP resistance.Exosomal miR-588 from M2 macrophages contributed to DDP resistance of gastric cancer cells.miR-588 promoted DDP-resistant gastric cancer cell growth in vivo.miR-588 was able to target cylindromatosis(CYLD)in gastric cancer cells.The depletion of CYLD reversed miR-588 inhibition-regulated cell proliferation and apoptosis of gastric cancer cells exposed to DDP.CONCLUSION In conclusion,we uncovered that exosomal miR-588 from M2 macrophages contributes to DDP resistance of gastric cancer cells by partly targeting CYLD.miR-588 may be applied as a potential therapeutic target for the treatment of gastric cancer.

MCP-1 and IL-4 encapsulated hydrogel particles with macrophages enrichment and polarization capabilities for systemic lupus erythematosus treatment

Macrophages play a pivotal role in systemic lupus erythematosus(SLE)therapy.Efforts have been made to develop multifunctional drug delivery systems capable of directing macrophage polarization.Here,we present a novel hyaluronic acid methacrylate(HAMA)hydrogel microparticle encapsulating multiple cytokines for SLE remission though enhancing macrophage functions.The HAMA microparticles loaded with monocyte chemotactic protein-1(MCP-1)and interleukin-4(IL-4)were fabricated by using a microfluidic technology.The released MCP-1 facilitates the aggregation of inflammatory macrophages,after which IL-4 induces the macrophage phenotype shift from inflammatory M1 to immune-protective M2,thus restoring immune balance.We have demonstrated in MRL/lpr mice that the hydrogel microparticles could improve their efficacy of intraperitoneal drug delivery,modulate immune function,and attenuate the disease symptoms.These results suggest that our proposed microparticles delivery platform has potential clinical value for treating autoimmune diseases.

M1-type microglia can induce astrocytes to deposit chondroitin sulfate proteoglycan after spinal cord injury

After spinal cord injury(SCI),astrocytes gradually migrate to and surround the lesion,depositing chondroitin sulfate proteoglycan-rich extracellular matrix and forming astrocytic scar,which limits the spread of inflammation but hinders axon regeneration.Meanwhile,microglia gradually accumulate at the lesion border to form microglial scar and can polarize to generate a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype.However,the effect of microglia polarization on astrocytes is unclear.Here,we found that both microglia(CX3 CR1^(+))and astrocytes(GFAP^(+))gathered at the lesion border at 14 days post-injury(dpi).The microglia accumulated along the inner border of and in direct contact with the astrocytes.M1-type microglia(i NOS^(+)CX3 CR1^(+))were primarily observed at 3 and 7 dpi,while M2-type microglia(Arg1^(+)CX3 CR1^(+))were present at larger numbers at 7 and 14 dpi.Transforming growth factor-β1(TGFβ1)was highly expressed in M1 microglia in vitro,consistent with strong expression of TGFβ1 by microglia in vivo at 3 and 7 dpi,when they primarily exhibited an M1 phenotype.Furthermore,conditioned media from M1-type microglia induced astrocytes to secrete chondroitin sulfate proteoglycan in vitro.This effect was eliminated by knocking down sex-determining region Y-box 9(SOX9)in astrocytes and could not be reversed by treatment with TGFβ1.Taken together,our results suggest that microglia undergo M1 polarization and express high levels of TGFβ1 at 3 and 7 dpi,and that M1-type microglia induce astrocytes to deposit chondroitin sulfate proteoglycan via the TGFβ1/SOX9 pathway.The study was approved by the Institutional Animal Care and Use Committee of Anhui Medical University,China(approval No.LLSC20160052)on March 1,2016.

Modulating poststroke inflammatory mechanisms: Novel aspects of mesenchymal stem cells, extracellular vesicles and microglia

Inflammation plays an important role in the pathological process of ischemic stroke,and systemic inflammation affects patient prognosis.As resident immune cells in the brain,microglia are significantly involved in immune defense and tissue repair under various pathological conditions,including cerebral ischemia.Although the differentiation of M1 and M2 microglia is certainly oversimplified,changing the activation state of microglia appears to be an intriguing therapeutic strategy for cerebral ischemia.Recent evidence indicates that both mesenchymal stem cells(MSCs)and MSC-derived extracellular vesicles(EVs)regulate inflammation and modify tissue repair under preclinical stroke conditions.However,the precise mechanisms of these signaling pathways,especially in the context of the mutual interaction between MSCs or MSC-derived EVs and resident microglia,have not been sufficiently unveiled.Hence,this review summarizes the state-ofthe-art knowledge on MSC-and MSC-EV-mediated regulation of microglial activity under ischemic stroke conditions with respect to various signaling pathways,including cytokines,neurotrophic factors,transcription factors,and microRNAs.

Regeneration-associated macrophages： a novel approach to boost intrinsic regenerative capacity for axon regeneration

Axons in central nervous system （CNS） do not regenerate spontaneously after injuries such as stroke and traumatic spinal cord iniury. Both intrinsic and extrinsic factors are responsible for the regeneration fail- ure, Although intensive research efforts have been invested on extrinsic regeneration inhibitors, the extent to which glial inhibitors contribute to the regeneration failure in viva still remains elusive. Recent exper- imental evidence has rekindled interests in intrinsic factors for the regulation of regeneration capacity in adult mammals. In this review, we propose that activating macrophages with pro-regenerative molecular signatures could be a novel approach for boosting intrinsic regenerative capacity of CNS neurons. Using a conditioning injury model in which regeneration of central branches of dorsal root ganglia sensory neu- rons is enhanced by a preceding injury to the peripheral branches, we have demonstrated that perineuronal macrophages surrounding dorsal root ganglia neurons are critically involved in the maintenance of en- hanced regeneration capacity. Neuron-derived chemokine （C-C motif） ligand 2 （CCL2） seems to mediate neuron-macrophage interactions conveying injury signals to perineuronal macrophages taking on a soley pro-regenerative phenotype, which we designate as regeneration-associated macrophages （RAMs）. Ma- nipulation of the CCL2 signaling could boost regeneration potential mimicking the conditioning injury, suggesting that the chemokine-mediated RAM activation could be utilized as a regenerative therapeutic strategy for CNS injuries.

Effects of Total Saponins from Dioscorea Nipponica Makino on Monosodium Urate-Induced M1-Polarized Macrophages through Arachidonic Acid Signaling Pathway:An in vitro Study

Objective To investigate and reveal the underlying mechanism of the effect of total saponins from Dioscoreae nipponica Makino(TSDN)on the arachidonic acid pathway in monosodium urate(MSU)crystal-induced M1-polarized macrophages.Methods M1 polarization of RAW264.7 cells were induced by 1µg/mL lipopolysaccharide(LPS).The methylthiazolyldiphenyl-tetrazolium bromide method was then used to screen the concentration of TSDN.MSU(500µg/mL)was used to induce the gouty arthritis model.Afterwards,10µg/L TSDN and 8µmol/L celecoxib,which was used as a positive control,were added to the above LPS and MSU-induced cells for 24 h.The mRNA and protein expressions of cyclooxygenase(COX)2,5-lipoxygenase(5-LOX),microsomal prostaglandin E synthase derived eicosanoids(mPGES)-1,leukotriene B(LTB)4,cytochrome P450(CYP)4A,and prostaglandin E_(2)(PGE_(2))were tested by real-time polymerase chain reaction and Western blotting,respectively.The enzyme-linked immunosorbent assay was used to test the contents of M1 markers,including inducible nitric oxid synthase(NOS)2,CD80,and CD86.Results TSDN inhibited the proliferation of M1 macrophages and decreased both the mRNA and protein expressions of COX2,5-LOX,CYP4A,LTB4,and PGE_(2)(P<0.01)while increased the mRNA and protein expression of mPGES-1(P<0.05 or P<0.01).TSDN could also significantly decrease the contents of NOS2,CD80,and CD86(P<0.01).Conclusion TSDN has an anti-inflammation effect on gouty arthritis in an in vitro model by regulating arachidonic acid signaling pathway.