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BackgroundLeptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca(2+) concentration ([Ca(2+)]i) elevation induced by infection can cause cell death, but [Ca(2+)]i changes and high [Ca(2+)]i-induced death of macrophages due to infection of Leptospira have not been previously reported.Methodology/principal findingsWe first used a Ca(2+)-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca(2+)]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca(2+)]i elevation was caused by both extracellular Ca(2+) influx through the purinergic receptor, P2X7, and Ca(2+) release from the endoplasmic reticulum, as seen by suppression of [Ca(2+)]i elevation when receptor-gated calcium channels were blocked or P2X7 was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3), which in turn induces intracellular Ca(2+) release from endoplasmic reticulum, with the Km of 199 µM and Kcat of 8.566E-5 S(-1). Secretion of L-PI-PLC from the spirochete into supernatants of leptospire-macrophage co-cultures and cytosol of infected macrophages was also observed by Western Blot assay. Lower [Ca(2+)]i elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca(2+)]i. Moreover, PI-PLCs (PI-PLC-β3 and PI-PLC-γ1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca(2+)]i increases induced apoptosis and necrosis of macrophages, while mild [Ca(2+)]i elevation only caused apoptosis.Conclusions/significanceThis study demonstrated that L. interrogans infection induced [Ca(2+)]i elevation through extracellular Ca(2+) influx and intracellular Ca(2+) release cause macrophage apoptosis and necrosis, and the LB361 gene product was shown to be a novel PI-PLC of L. interrogans responsible for the [Ca(2+)]i elevation.

Background: The dynamic interaction between cancer cells and tumour-associated macrophages (TAMs) in the hypoxic tumour microenvironment (TME) is an active barrier to the effector arm of the antitumour immune response. Cancer-secreted exosomes are emerging mediators of this cancer-stromal cross-talk in the TME; however, the mechanisms underlying this interaction remain unclear., Methods: Exosomes were isolated with ExoQuick exosome precipitation solution. The polarizing effect of TAMs was evaluated by flow cytometry, western blot analysis, immunofluorescence staining and in vitro phagocytosis assays. Clinical cervical cancer specimens and an in vivo xenograft model were also employed., Results: Our previous study showed that hypoxia increased the expression of ZEB1 in cervical squamous cell carcinoma (CSCC) cells, which resulted in increased infiltration of TAMs. Here, we found that hypoxia-induced ZEB1 expression is closely correlated with CD47-SIRPα axis activity in CSCC, which enables cancer cells to evade phagocytosis by macrophages and promotes tumour progression. ZEB1 was found to directly activate the transcription of the CD47 gene in hypoxic CSCC cells. We further showed that endogenous ZEB1 was characteristically enriched in hypoxic CSCC cell-derived exosomes and transferred into macrophages via these exosomes to promote SIRPα + TAM polarization. Intriguingly, exosomal ZEB1 retained transcriptional activity and reprogrammed SIRPα + TAMs via activation of the STAT3 signalling pathway in vitro and in vivo. STAT3 inhibition reduced the polarizing effect induced by exosomal ZEB1. Knockdown of ZEB1 increased the phagocytosis of CSCC cells by macrophages via decreasing CD47 and SIRPα expression., Conclusions: Our results suggest that hypoxia-induced ZEB1 promotes immune evasion in CSCC by strengthening the CD47-SIRPα axis. ZEB1-targeted therapy in combination with CD47-SIRPα checkpoint immunotherapy may improve the outcomes of CSCC patients in part by disinhibiting innate immunity., (© 2023. The Author(s).)

Background: Fibrotic scar formation and inflammation are characteristic pathologies of spinal cord injury (SCI) in the injured core, which has been widely regarded as the main barrier to axonal regeneration resulting in permanent functional recovery failure. Pericytes were shown to be the main source of fibroblasts that form fibrotic scar. However, the mechanism of pericyte-fibroblast transition after SCI remains elusive., Methods: Fibrotic scarring and microvessels were assessed using immunofluorescence staining after establishing a crush SCI model. To study the process of pericyte-fibroblast transition, we analyzed pericyte marker and fibroblast marker expression using immunofluorescence. The distribution and cellular origin of platelet-derived growth factor (PDGF)-BB were examined with immunofluorescence. Pericyte-fibroblast transition was detected with immunohistochemistry and Western blot assays after PDGF-BB knockdown and blocking PDGF-BB/PDGFRβ signaling in vitro. Intrathecal injection of imatinib was used to selectively inhibit PDGF-BB/PDGFRβ signaling. The Basso mouse scale score and footprint analysis were performed to assess functional recovery. Subsequently, axonal regeneration, fibrotic scarring, fibroblast population, proliferation and apoptosis of PDGFRβ + cells, microvessel leakage, and the inflammatory response were assessed with immunofluorescence., Results: PDGFRβ + pericytes detached from the blood vessel wall and transitioned into fibroblasts to form fibrotic scar after SCI. PDGF-BB was mainly distributed in the periphery of the injured core, and microvascular endothelial cells were one of the sources of PDGF-BB in the acute phase. Microvascular endothelial cells induced pericyte-fibroblast transition through the PDGF-BB/PDGFRβ signaling pathway in vitro. Pharmacologically blocking the PDGF-BB/PDGFRβ pathway promoted motor function recovery and axonal regeneration and inhibited fibrotic scar formation. After fibrotic scar formation, blocking the PDGFRβ receptor inhibited proliferation and promoted apoptosis of PDGFRβ + cells. Imatinib did not alter pericyte coverage on microvessels, while microvessel leakage and inflammation were significantly decreased after imatinib treatment., Conclusions: We reveal that the crosstalk between microvascular endothelial cells and pericytes promotes pericyte-fibroblast transition through the PDGF-BB/PDGFRβ signaling pathway. Our finding suggests that blocking the PDGF-BB/PDGFRβ signaling pathway with imatinib contributes to functional recovery, fibrotic scarring, and inflammatory attenuation after SCI and provides a potential target for the treatment of SCI., (© 2022. The Author(s).)

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 (CX3CR1 + ) 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 (iNOS + CX3CR1 + ) were primarily observed at 3 and 7 dpi, while M2-type microglia (Arg1 + CX3CR1 + ) 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., Competing Interests: None

Purpose: Clinical research suggests that transcranial direct current stimulation (tDCS) at bilateral supraorbital foramen and inferior orbital rim and nose intersections may facilitate rehabilitation after stroke. However, the underlying neurobiological mechanisms of tDCS remain poorly understood, impeding its clinical application. Here, we investigated the effect of tDCS applied after stroke on neural cells., Materials and Methods: Middle cerebral arterial occlusion (MCAO) reperfusion was induced in rats. Animals with comparable infarcts were randomly divided into MCAO group and MCAO + tDCS group. Recovery of neurological function was assessed behaviorally by modified neurological severity score (mNSS). Ischemic tissue damage verified histologically by TTC and HE staining. Immunohistochemical staining, real-time qPCR, and western blot were applied to determine the changes of neural cells in ischemic brains., Results: The results reveal that tDCS treated by multilead brain reflex instrument can promote the recovery of neurological function, remarkably reduce cerebral infarct volume, promote brain tissue rehabilitation, and can effectively inhibit astrocytosis and enhance neuronal survival and synaptic function in ischemic brains., Conculsions: Our study suggests that tDCS treated by multilead brain reflex instrument could be prospectively developed into a clinical treatment modality.

We previously demonstrated that overexpression of tropomyosin receptor kinase A (TrkA) promotes the survival and Schwann cell-like differentiation of bone marrow stromal stem cells in nerve grafts, thereby enhancing the regeneration and functional recovery of the peripheral nerve. In the present study, we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts. Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA, TrkA-shRNA or the respective control. The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect. Then, 8 weeks after surgery, hematoxylin and eosin staining showed that compared with the control groups, the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged, whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group. Western blot assay showed that compared with the control groups, the TrkA overexpressing group had higher expression of the myelin marker, myelin basic protein and the axonal marker neurofilament 200. The TrkA overexpressing group also had higher levels of various signaling molecules, including TrkA, pTrkA (Tyr490), extracellular signal-regulated kinases 1/2 (Erk1/2), pErk1/2 (Thr202/Tyr204), and the anti-apoptotic proteins Bcl-2 and Bcl-xL. In contrast, these proteins were downregulated, while the pro-apoptotic factors Bax and Bad were upregulated, in the TrkA-shRNA group. The levels of the TrkA effectors Akt and pAkt (Ser473) were not different among the groups. These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway. All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University, China in December 2014 (approval No. AEWC-2014-001219)., Competing Interests: None

To investigate the effects of Liu Tea extracts(LTE) on proliferation, apoptosis and drug sensitivity of drug-resistant gastric cancer cell BGC823/5-FU. MTT assay was used to analyze effect of LTE on cell growth and sensitivity chemotherapeutic drugs, and synergistic effect of the combination of LTE with 5-FU on BGC823/5-FU cells. Combination index (CI) was calculated by CompuSyn. Cell apoptosis was measured by flow cytometry (FCM). Protein expressions of P-gp, Bcl-2, Bax and Caspase-3 (17KD) were detected by Western blot at different concentrations of LTE in BGC823/5-FU cells (100, 200, 400 mg•L⁻¹). The results showed that LTE had an inhibitory effect on growth of BGC823/5-FU cell in a dose-time-dependent manner and significantly reduced IC₅₀ of 5-FU, CDDP, PTX and ADM to BGC823/5-FU cells(P<0.05), indicating it could reverse tolerance of drug resistant cells to chemotherapeutic drugs, with reversion multiples of 2.35, 1.68, 1.96, 0.52. The combination of LTE with 5-FU had positive synergistic effect on the BGC-823 cell line. FCM assay suggested that LTE could induce BGC823/5-FU apoptosis. The apoptosis rate was up to 46.2% when the cells were treated with 800 mg•L⁻¹ LTE after 24 h(P<0.01). According to the protein detection results, with the increase in concentration of LTE, the protein expression of Bcl-2 was gradually decreased(P<0.01), the expression of Bax and Caspase-3 were extremely increased(P<0.01), with statistical significance in difference(P<0.01) but no difference in the expression of P-gp between experiment group and control group. LTE can inhibit the growth of drug-resistant human gastric cancer cell BGC823/5-FU and reverse its chemotherapeutic tolerance to some extent. Inhibition of antiapoptotic proteins, activation of proapoptotic proteins and induction of apoptosis of resistant cells may be its main mechanisms., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Objective: To explore the potential precursors of the anthraquinone metabolites from Rheum tanguticum and preliminanly identify the synthesis pathway thereof., Methods: Sterile seedlings sprouted from the seeds of Rheum tanguticum were chosen as materials for inducing callus. The effects of different precursors and feeding duration on the callus of Rheum tanguticum and the anthraquinone yield in adult rheum were studied., Results: The greatest improvement of anthraquinone yield was achieved by acetic acid, increasing 43. 9% for the callus and 45. 8% in the adult rheum; the second greatest improvement was achieved by malonic acid, increasing 15. 8% for the callus and only 3. 6% in the adult rheum. The yield of anthraquinone was not influenced significantly by benzoic acid and p-benzoquinone, and in contrast, was inhibited in some degree by shikimic acid and α-ketoglutaric acid. A suitable feeding duration was 36 h, which worked well for the effects of precursors., Conclusion: The precursor for synthesis of anthraquinone metabolites from Rheum tan- guticum is acetic acid, which improves the yields of callus and anthraquinone in adult rheum, concluding that the anthraquinone metabolites are synthesized via polyketone pathway.

Objective: To establish the approach to detecting two biovars of Ureaplasma urealyticum (Uu) in human semen and to investigate the relationship between the two biovars of Uu infection and the quality of human semen., Methods: Based on the 16S-23S rRNA intergenic spacer region, three pairs of primers were designed, the species specific primer and two biovars primers (Parvo primer and T960 primer). The two biovars of Uu were detected in the semen from 949 men by semen culture and PCR assay. Meanwhile, semen routine analyses were performed., Results: In the 949 subjects, 199 were Uu positive both in Uu liquid culture and PCR assay (199/949, 21.1%), of which 136 (136/199, 68.3%) were Parvo biovar, 54 (54/199, 27.1%) T960 biovar, and 9 (9/199, 4.5%) both Parvo and T960 biovars. Compared with the Parvo and the negative groups, human sperm viability was significantly decreased (P < 0.05 ) in the Uu T960 infection group. The difference of sperm motility and density had no statistic significance., Conclusion: A significant correlation has been found between Uu T960 biovar infection and human sperm viability