A novel small-molecular CCR5 antagonist promotes neural repair after stroke.

Chemokine receptor 5 (CCR5) is one of the main co-receptors of HIV-1, and has been found to be a potential therapeutic target for stroke. Maraviroc is a classic CCR5 antagonist, which is undergoing clinical trials against stroke. As maraviroc shows poor blood-brain barrier (BBB) permeability, it is of interest to find novel CCR5 antagonists suitable for neurological medication. In this study we characterized the therapeutic potential of a novel CCR5 antagonist A14 in treating ischemic stroke mice. A14 was discovered in screening millions compounds in the Chemdiv library based on the molecular docking diagram of CCR5 and maraviroc. We found that A14 dose-dependently inhibited the CCR5 activity with an IC50 value of 4.29 µM. Pharmacodynamic studies showed that A14 treatment exerted protective effects against neuronal ischemic injury both in vitro and vivo. In a SH-SY5Y cell line overexpressing CCR5, A14 (0.1, 1 µM) significantly alleviated OGD/R-induced cell injury. We found that the expression of CCR5 and its ligand CKLF1 was significantly upregulated during both acute and recovery period in focal cortical stroke mice; oral administration of A14 (20 mg·kg-1·d-1, for 1 week) produced sustained protective effect against motor impairment. A14 treatment had earlier onset time, lower onset dosage and much better BBB permeability compared to maraviroc. MRI analysis also showed that A14 treatment significantly reduced the infarction volume after 1 week of treatment. We further revealed that A14 treatment blocked the protein-protein interaction between CCR5 and CKLF1, increasing the activity of CREB signaling pathway in neurons, thereby improving axonal sprouting and synaptic density after stroke. In addition, A14 treatment remarkably inhibited the reactive proliferation of glial cells after stroke and reduced the infiltration of peripheral immune cells. These results demonstrate that A14 is a promising novel CCR5 antagonist for promoting neuronal repair after ischemic stroke. A14 blocked the protein-protein interaction between CKLF1 and CCR5 after stroke by binding with CCR5 stably, improved the infarct area and promoted motor recovery through reversing the CREB/pCREB signaling which was inhibited by activated CCR5 Gαi pathway, and benefited to the dendritic spines and axons sprouting.

A breakdown of metabolic reprogramming in microglia induced by CKLF1 exacerbates immune tolerance in ischemic stroke.

Ischemic stroke is characterized by the presence of reactive microglia. However, its precise involvement in stroke etiology is still unknown. We used metabolic profiling and showed that chemokine like factor 1 (CKLF1) causes acute microglial inflammation and metabolic reprogramming from oxidative phosphorylation to glycolysis, which was reliant on the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR)-hypoxia inducible factor 1α (HIF-1α) signaling pathway. Once activated, microglia enter a chronic tolerant state as a result of widespread energy metabolism abnormalities, which reduces immunological responses, including cytokine release and phagocytosis. Metabolically dysfunctional microglia were also found in mice using genome-wide RNA sequencing after chronic administration of CKLF1, and there was a decrease in the inflammatory response. Finally, we showed that the loss of CKLF1 reversed the defective immune response of microglia, as indicated by the maintenance its phagocytosis to neutrophils, thereby mitigating the long-term outcomes of ischemic stroke. Overall, CKLF1 plays a crucial role in the relationship between microglial metabolic status and immune function in stroke, which prepares a potential therapeutic strategy for ischemic stroke.

A rabies virus-based toolkit for efficient retrograde labeling and monosynaptic tracing.

Analyzing the structure and function of the brain's neural network is critical for identifying the working principles of the brain and the mechanisms of brain diseases. Recombinant rabies viral vectors allow for the retrograde labeling of projection neurons and cell type-specific trans-monosynaptic tracing, making these vectors powerful candidates for the dissection of synaptic inputs. Although several attenuated rabies viral vectors have been developed, their application in studies of functional networks is hindered by the long preparation cycle and low yield of these vectors. To overcome these limitations, we developed an improved production system for the rapid rescue and preparation of a high-titer CVS-N2c-ΔG virus. Our results showed that the new CVS-N2c-ΔG-based toolkit performed remarkably: (1) N2cG-coated CVS-N2c-ΔG allowed for efficient retrograde access to projection neurons that were unaddressed by rAAV9-Retro, and the efficiency was six times higher than that of rAAV9-Retro; (2) the trans-monosynaptic efficiency of oG-mediated CVS-N2c-ΔG was 2-3 times higher than that of oG-mediated SAD-B19-ΔG; (3) CVS-N2c-ΔG could delivery modified genes for neural activity monitoring, and the time window during which this was maintained was 3 weeks; and (4) CVS-N2c-ΔG could express sufficient recombinases for efficient transgene recombination. These findings demonstrate that new CVS-N2c-ΔG-based toolkit may serve as a versatile tool for structural and functional studies of neural circuits.

The versatile role of TREM2 in regulating of microglia fate in the ischemic stroke.

Microglia are the earliest activated and the longest lasting immune cells after stroke, and they participate in almost all the pathological reactions after stroke. However, their regulatory mechanism has not been fully elucidated. Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor that is mainly expressed in microglia of the central nervous system. The receptor plays an important role in regulating microglia energy metabolism and phenotypic transformation. At present, TREM2 has been developed as a potential target for AD, coronary atherosclerosis and other diseases. However, TREM2 does not provide a systematic summary of the functional transformation and intrinsic molecular mechanisms of microglia after stroke. In this paper, we have summarized the functional changes of TREM2 in microglia after stroke in recent years, and found that TREM2 has important effects on energy metabolism, phagocytosis and anti-inflammatory function of microglia after stroke, suggesting that TREM2 is a potential therapeutic target for the treatment of stroke.

Flavonoid-enriched extract from Millettia speciosa Champ prevents obesity by regulating thermogenesis and lipid metabolism in high-fat diet-induced obese C57BL/6 mice.

Millettia speciosa (M. speciosa) Champ is a medicinal and edible plant. The roots are rich in flavonoids, which possess multiple biological activities, including lipid-lowering effects. This study aimed to explore the effect of flavonoid-enriched extract from M. speciosa (FMS) on obesity. The UPLC-Q-TOF-MS analysis and chromatographic analysis were adopted to identify flavonoid compounds in FMS. Male C57BL/6J mice were fed with a high-fat diet for 3 months and were then treated with FMS (50 or 100 mg/kg/d) or Orlistat (10 mg kg-1 d-1) for another 8 weeks. A total of 35 flavonoids were identified in the extract of M. speciosa root. FMS reduced body weight gain, liver weight gain, white adipose tissue, lipid accumulation, and blood glucose. The levels of TG, ALT, AST, and inflammatory-related adipokines (TNF-α and IL-6) in serum were also reduced by FMS. In addition, FMS promoted thermogenesis in brown adipose tissue and induced the activation of lipolysis, fatty acid oxidation, and oxidative phosphorylation in white adipose tissues. In summary, long-term administration of FMS could ameliorate high-fat diet-induced obesity by stimulating adipose thermogenesis and lipid metabolism.

The Role of AMPARs Composition and Trafficking in Synaptic Plasticity and Diseases.

AMPA receptors are tetrameric ionic glutamate receptors, which mediate 90% fast excitatory synaptic transmission induced by excitatory glutamate in the mammalian central nervous system through the activation or inactivation of ion channels. The alternation of synaptic AMPA receptor number and subtype is thought to be one of the primary mechanisms that involve in synaptic plasticity regulation and affect the functions in learning, memory, and cognition. The increasing of surface AMPARs enhances synaptic strength during long-term potentiation, whereas the decreasing of AMPARs weakens synaptic strength during the long-term depression. It is closely related to the AMPA receptor as well as its subunits assembly, trafficking, and degradation. The dysfunction of any step in these precise regulatory processes is likely to induce the disorder of synaptic transmission and loss of neurons, or even cause neuropsychiatric diseases ultimately. Therefore, it is useful to understand how AMPARs regulate synaptic plasticity and its role in related neuropsychiatric diseases via comprehending architecture and trafficking of the receptors. Here, we reviewed the progress in structure, expression, trafficking, and relationship with synaptic plasticity of AMPA receptor, especially in anxiety, depression, neurodegenerative disorders, and cerebral ischemia.

[Advances of Akkermansia muciniphila in regulating host functions].

Akkermansia muciniphila, abbreviated as AKK and found in 2004, is an oval-shaped gram-negative bacterium isolated from a human feal. A. muciniphila is widely present in the intestinal tract of human. Its specialization in mucin degradation makes it a key organism at the mucosal interface between the lumen and host cells. More and more studies have shown that it can play the role of probiotics. Notably, declined levels of A. muciniphila have been observed in patients with diabetes, liver disease, cardiovascular disease, inflammatory bowel disease, neurodegenerative diseases, etc. In addition, A. muciniphila combined with traditional Chinese medicine, exhibited higher effect on regulating host functions, but the underlying mechanism was still unclear, requiring further in-depth research. Therefore, the aims of this review are to summarize the main effects of A. muciniphila on host health and its relationship with traditional Chinese medicine, summarize the main problems, and provide a reference for the further research of A. muciniphila and traditional Chinese medicine.

Coptisine from Rhizoma coptidis exerts an anti-cancer effect on hepatocellular carcinoma by up-regulating miR-122.

With increasing incidence and mortality, hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. In this study, microRNA-122 (miR-122) mimics and relevant control oligonucleotides were transfected into HepG2 cells in vitro, followed by coptisine (COP) and sorafenib treatments. Cell proliferation, migration, and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay, wound-healing assay, Hoechst 33258 staining and flow cytometry, respectively. Histopathology and miR-122 were analyzed by haemotoxylin and eosin (H&E) staining and real-time RT-PCR, respectively; whereas, the relevant protein expressions were detected by western blot. In vivo, COP enhanced the expression of miR-122 by 160% compared to control in male BALB/c nude mice; COP not only protected the liver morphology but also showed a significant anti-cancer effect. Further, there was no remarkable difference between the tumor weights in the COP and sorafenib groups, but there was a striking difference to the tumor control group (p < 0.05). Hence, COP inhibited the proliferation, migration and promoted apoptosis of HCC cells; moreover, it inhibited the tumor growth in nude mice by up-regulating the expression of miR-122.

Protective effect of Coptisine from Rhizoma Coptidis on LPS/D-GalN-induced acute liver failure in mice through up-regulating expression of miR-122.

Coptisine (COP), one of the main active ingredients of Rhizoma Coptidis, reportedly has anti-inflammatory, anti-colon cancer properties, but it remains elusive whether COP owns hepatoprotective activity. Mice were pretreated with COP for 7d prior to lipopolysaccharide/d-galactosamine (LPS/D-GalN) administration to detect the hepatic protective effects of COP. The mechanism was explored in using HepG2 cells with low level of miR-122 and LO2 cells with high level of miR-122, combining with miR-122 agomir transfection by means of detecting the expression of miR-122 and proteins, clinical index and apoptosis. COP ameliorated the LPS/D-GalN-induced liver failure by lowering serum levels of ALT and AST, raising hepatic GSH and SOD levels, and maintaining the morphology of hepatocytes, along with an increase in miR-122 expression in mice. The results in vitro indicated that, after miR-122 mimic administration, the alone treatment of COP and the co-treatment of COP and LPS transfection obviously promoted the apoptosis of HepG2, which was increased by 152.67% and 113.97% compared with NC (P < 0.05 vs NC). LPS significantly induced the apoptosis of L02 cells, but COP treatment attenuated that of L02 cells. Further analysis showed that COP increased the miR-122 level and the expression of Bax, cleaved-casp3 and decreased Bcl-2, Bcl-xL in LPS-treated HepG2 cells. COP increased the miR-122 level but decreased the expression of TLR4, Bcl-2, Bcl-xL in LPS-treated L02 cells. COP attenuated LPS/D-GalN-induced ALF by up-regulating the level of miR-122, synergistically promoting apoptosis, and suggesting COP which showed a potential protective effect on ALF.

[Preparation, characterization and preliminary application of monoclonal antibody against human mu chain].

AIM: To prepare monoclonal antibody (mAb) against human mu chain with high titer and establish a capture ELISA for early serological diagnosis of infectious diseases. METHODS: BALB/c mice were immunized with human IgM. Hybridoma cell line which could stably secret the mAb to human IgM was established by routine cell fusion technique. mAb's characteristics (titer, Ig subclass, specificity and relative affinity) were identified by indirect ELISA and Western blot, respectively. A capture ELISA was established by using purified mAb to capture specific IgM for early diagnosis of Japanese encephalitis. RESULTS: One hybridoma cell line 2E5 stably secreting mAb against human IgMmu chain was obtained. The titer of ascites of the mAb was 1 x 10(-6) and the Ig subclass was IgG1(kappa). Relative affinity of 2E5 was 1 x 10 (-5). Western blot analysis showed that mAb 2E5 reacted specifically to mu chain. Both sensitivity and specificity of the capture ELISA in detecting specific IgM in Japanese encephalitis patients sera were high. CONCLUSION: mAb 2E5 against human mu chain was prepared successfully, and a capture ELISA for early serological diagnosis of Japanese encephalitis was set up.

[Expression of Japanese encephalitis virus E protein in methylotrophic yeast Pichia pastoris].

AIM: To express Japanese encephalitis virus (JEV) E protein in methylotrophic yeast Pichia pastoris. METHODS: The gene coding for JEV E protein was sub-cloned into vector pPIC9k. The constructed plasmid was transformed into yeast SMD1168 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of E protein in yeast was induced by the addition of methanol and analyzed by SDS-PAGE and Western blot. RESULTS: The protein was produced at a yield of 50 mg per litre of culture. Owing to heterogeneous glycosylation, relative molecular mass (M(r)) of the expressed E protein was sized about 113 000 and 78 000. CONCLUSION: JEV E protein was expressed successfully in yeast Pichia pastoris, which should be useful for the production of diagnostic reagents and genetically engineered vaccine of JEV.

[Analysis on patial amino acid sequence of a novel glycoprotein binding to antiherpes simplex virus monoclonal antibody CHA9].

AIM: To acquire the characteristic amino acid sequence of a novel glycoprotein of herpes simplex virus (HSV), so as to localize gene encoding the novel glycoprotein accurately. METHODS: A 12-mer phage peptide library was screened for 3 rounds by using biotinylated mAb CHA9 against new glycoprotein g30k of HSV-2. Positive phage clones were detected by ELISA. 10 positive clones were selected randomly for sequencing. Sequence alignment and hydrophobic cluster analysis were carried out. RESULTS: Most of the sequences of 10 positive phage clones were highly homologous with a core-PH/KHXHXGS-. Phages containing this motif could react specifically with the mAb CHA9 and not cross-react with other IgG. Hydrophobic cluster analysis showed that the peptide was likely to form an epitope. CONCLUSION: We obtained a characteristic short peptide which shows some characters of g30K amino acid sequence. It provides valuable clue for prediction of the open reading frame of g30K gene and will be useful to explore biological characteristics of the new glycoprotein.