Objective: To investigate whether rapamycin treatment starting at 24 h after cerebral ischemia/reperfusion(I/R) has protective effect on brain injury in rats. Methods: The rat I/R model was established by middle cerebral artery occlusion according to Longa's method. A total of 104 Sprague Dawley rats were randomly divided into sham group, model group, and rapamycin-treated groups (6 h or 24 h after modeling). Neurological function was assessed with neurological severity score (NSS). Triphenyl tetrazolium chloride (TTC) staining and Fluoro-Jade B (FJB) staining were used to examine the infarct volume and neuronal apoptosis, respectively. The expression of p-S6 protein in mTOR signaling pathway was detected by Western blot analysis. Results: Compared with sham group, NSS of the model group was significantly increased and TTC staining indicated obvious infarct area (all P < 0.01). Furthermore, significantly increased number of FJB-positive cells and p-S6 expression in the penumbra area were shown in the model group (all P < 0.01). Compared with the model group, both rapamycin-treated groups demonstrated decreased NSS, infarction volume and FJB positive cells as well as p-S6 expression in the penumbra area (P < 0.05 or P < 0.01). There was no significant difference between the groups of rapamycin administrated 6 h and 24 h after modeling (all P > 0.05). Conclusion: Rapamycin treatment starting at 24 h after I/R exhibits protective effect on brain injury in rats.

Objective: To investigate the effects of Honokiol on cognitive function in mice with epilepsy. Methods: Kainic acid (38 mg/kg) was intraperitoneally injected in 5 weeks old male ICR mice to induce epilepsy. Honokiol at dose of 3, 10, 30 mg/kg was given to epilepic mice by intraperitoneal injection for 10 days. Fluoro-Jade B staining was used to assess neuronal death; Morris water maze and Y maze tests were used to measure cognitive function such as learning and memory; Western blot was performed to detect the expression of acetylated superoxide dismutase (SOD), microtubule associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) and P62 in hippocampus tissue; thiobarbituric acid and WST-1 methods were used to detect malondialdehyde (MDA) and SOD. Results: Compared with control group, the levels of acetylated-SOD, MDA, LC3-Ⅱ, P62 and neuronal death increased, cognitive function and SOD decreased in model group (P < 0.05 or P < 0.01). Honokiol at the dose of 10 mg/kg and 30 mg/kg decreased SOD acetylation, MDA content, expression of LC3-Ⅱ and P62, as well as neuronal death, and the cognitive function was improved (P < 0.05 or P < 0.01), especially in 30 mg/kg Honokiol group. Conclusion: Honokiol alleviates oxidative stress and autophagy degradation disorder, decreases neuronal death, and therefore improves cognitive function in epilepsy mice.

Objective: To investigate the efficacy of brain-targeted rapamycin (T-Rap) in treatment of epilepsy in rats. Methods: Rapamycin nanoparticles targeting brain were prepared. The epilepsy model was induced by injection of pilocarpine in rats. The rats with pilocarpine-induced epilepsy were treated with rapamycin (Rap group) or brain-targeted rapamycin (T-Rap group). Seizure activity was observed by electroencephalography; the effect on mTOR signaling pathway was detected by Western blot; neuronal death and moss fiber sprouting were analyzed by Fluoro-Jade B (FJB) and Timm's staining, respectively. Results: Electroencephalography showed that both preparation of rapamycin significantly reduced the frequency of spontaneous seizures in rats, and the effect of T-Rap was stronger than that of conventional rapamycin (P < 0.05). Western blot showed that the phosphorylation levels of S6K and S6 in T-Rap group were lower than those in Rap group (all P < 0.05), indicating that T-Rap had a stronger inhibitory effect on mTOR signaling pathway. FJB staining showed that T-Rap significantly decreased neuronal death, but there was no significant difference as compared with Rap group. Timm's staining showed that both preparations of rapamycin significantly reduced the germination of mossy fibers, while the effect of T-Rap was more pronounced than Rap group (P < 0.05). The inhibition of body weight gain of T-Rap group was less than that of Rap group (P < 0.05). Conclusion: T-Rap has a better therapeutic effect on epilepsy than conventional rapamycin with a less adverse effects in rats.

Objective: To investigate the effect of rapamycin on Parkinson's disease (PD) and its underlying mechanism in mice. Methods: Sixty SPF adult male C57BL/6 mice were randomly divided into control group, model group and treatment group. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine(MPTP) was used to induce Parkinson's disease in model group and treatment group. All mice were trained to cross the runway and were subjected to computer-assisted CatWalk. The numbers of tyrosine hydroxylase positive (TH⁺) neurons in the substantia nigra (SN) were assessed by unbiased stereology using the optical fractionator method; protein expression was detected by Western blot analysis; and glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by spectrophotometry. Results: In the model group, a decrease in stride rate and an increase in variation of stance and swing were noted by CatWalk system (P < 0.05 or P < 0.01); the numbers of TH⁺ neurons decreased (P < 0.01); expression of p-Akt, p-S6K, p-S6 and p-ULK increased (all P < 0.01); LC3-Ⅱ/Ⅰ ratio decreased (P < 0.01); MDA level was elevated while the levels of SOD and GSH-PX were reduced (all P < 0.01). Compared with the model group, after treated with rapamycin, the abnormal behavior including the stride length, variation of stance and swing and step patterns induced by MPTP were all improved (P < 0.05 or P < 0.01); the numbers of TH⁺ neurons increased (P < 0.05); the expression of p-Akt, p-S6K, p-S6 and p-ULK was suppressed (all P < 0.01); the LC3-Ⅱ/Ⅰ ratio was upregulated (P < 0.05); MDA level decreased while the levels of GSH-Px and SOD increased (all P < 0.01). Conclusion: Rapamycin inhibits the activation of mTOR pathway, which contributes to protect against the loss of dopaminergic neurons and provide behavioral improvements in mice with Parkinson's disease. These results are partially related to the ability of rapamycin in inducing autophagy and reducing oxidative stress.

Objective: To investigate the effect of osthole on the expression of amyloid precursor protein (APP) in Alzheimer's disease (AD) cell model and its mechanism. Methods: The SH-SY5Y cell with over expression of APP was established by transfection by liposome 2000. The cells were treated with different concentrations of osthole, and the cell viability was determined by MTT and lactate dehydrogenase (LDH) assay. The differentially expressed miRNAs with and without osthole treatment were detected by miRNA array, and the target genes binding to the differentially expressed miRNAs were identified and verified by databases and Cytoscape. After the inhibitor of the differentially expressed miRNA was transduced into cells, the changes of APP and amyloid β (Aβ) protein were determined by immunofluorescence cytochemistry, and the mRNA expression of APP was determined by RT-PCR. Results: The AD cell model with over expression of APP was established successfully. The results of MTT and LDH assay showed that osthole had a protective effect on cells and alleviated cell damage. miR-101a-3p was identified as the differentially expressed miRNA, which was binding to the 3'-UTR of APP. Compared with APP group, the expression of APP and Aβ protein and APP mRNA increased in the miR-101a-3p inhibitor group (all P < 0.01), while the expression of APP and Aβ protein and APP mRNA decreased in the cells with osthole treatment (all P < 0.01). Conclusion: Osthole inhibits the expression of APP by up-regulating miR-101a-3p in AD cell model.

Objective: To investigate the effect of curcumin on dopamine neurons in Parkinson's disease (PD) and its mechanism. Methods: SH-SY5Y human neuroblastoma cells were treated with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to establish the PD cell model. The model cells were treated with curcumin and/or autophagy inhibitor 3-MA. After 48 h of drug treatment, the number of surviving dopamine neurons was detected by tyrosine hydroxylase immunofluorescence method. Western blotting was used to detect protein expression of α-Synuclein (α-Syn), transcription factor EB (TFEB) and autophagy-related proteins lysosome-associated membrane protein 2A (LAMP2A) and microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ); RT-PCR was used to detect mRNA expression of α-Syn. Results: Compared with MPTP model group, curcumin increased the number of surviving dopamine neurons(P < 0.01), decreased both protein expression and mRNA expression of α-Syn (all P < 0.01), and increased protein expression of TFEB, LAMP2A and LC3-Ⅱ (all P < 0.01). When curcumin and 3-MA were given concurrently, the number of surviving dopamine neurons, protein expression of TFEB, LAMP2A and LC3-Ⅱ increased (P < 0.05 or P < 0.01), and both protein expression and mRNA expression of α-Syn decreased (P < 0.05 or P < 0.01) compared with MPTP model group; but the number of surviving dopamine neurons and protein expression of LAMP2A and LC3-Ⅱ decreased compared with curcumin group (all P < 0.05). Conclusion: Curcumin exerts protective effect on dopamine neurons in PD, which may be associated with enhancing autophagy and promoting the clearance of α-Syn.

Objective: To investigate the effect of G protein-coupled receptor 17 (GPR17) on hypoxia injury in retinal ganglion cells in vitro. Methods: CoCl₂ (400 μmol/L) was used to induce hypoxic injury in RGC-5 cells. The expression of GPR17 and the effect of GPR17 ligands were investigated, and the role of GPR17 in hypoxia injury was further studied by transfection of RGC-5 cells with GPR17 small interfering RNA (siRNA). The cell viability was determined by MTT and the cell apoptosis rate was detected by flow cytometry analysis. The expression of GPR17 mRNA was determined with RT-PCR. Results: mRNA expressions of GPR17 in RGC-5 cells with and without CoCl₂ treatment were 0.36±0.05 and 0.26±0.08(P < 0.01). Compared with hypoxia without any treatment, pretreatment with GPR17 agonists (LTD₄, UDP, UDP-G) significantly reduced cell viability (the survival rates of cells decreased by 29.6%, 31.8% and 33.9%, all P < 0.01), while the effect of GPR17 antagonist (cangrelor) was the opposite (the survival rates of cells increased by 33.2%, P < 0.01). Transfection with GPR17 SiRNA inhibited hypoxia-induced up-expression of GPR17 mRNA (P < 0.01)and reduced cell apoptosis[rates of cell apoptosis were(39.73±2.06)%, (42.50±3.64)% and (24.98±2.16)% for blank control, NC siRNA and GPR17 siRNA groups, P < 0.01]. Conclusion: GPR17 may mediate hypoxia injury in RGC-5 cells, while the knockdown of GPR17 can reduce the hypoxia injury.

Astrocytes are the most abundant cells in the central nervous system and play significant roles in normal brain. With cerebral infarction, astrocytes are activated as reactive astrocytes and form glial scars, which play an essential part in brain injury. According to their roles in neuroprotection after cerebral infarction, regulation of scar formation, nerve regeneration, maintenance of blood-brain barrier, promotion of angiogenesis and immune response, scholars have proposed a variety of therapeutic strategies based on targeting astrocytes. This article reviews the research progress on the changes in astrocyte signaling pathways before and after cerebral infarction and the related therapeutic strategies.

Objective: To investigate the anti-inflammatory effect and mechanisms of interleukin-35 (IL-35) in inflammatory bowel disease. Methods: BALB/c mice were divided into three groups with 10 mice in each group:control group, model group (oral administration of 4% glucan sodium sulfate for 7 d) and IL-35-treated group (oral administration of 4% glucan sodium sulfate for 7 d, intraperitoneal injection of 2 μg IL-35 at d2-5). Disease activity index (DAI) was scored every day. After 7 d, the mice were sacrificed, and the serum and intestinal tissue samples were collected. The gross morphology of the colon was observed; HE staining was used to observe the pathological changes of colon tissue; flow cytometry was employed to detect the change of macrophage polarization ratio in colon tissue; the mRNA expression levels of cytokines IL-6, TNF-α, IFN-γ, IL-10 and SHIP1 in colon tissue were determined by real-time quantitative RT-PCR; the expression and distribution of SHIP1 in colon tissue was measured by immunohistochemistry; Western blotting was adopted to detect the expression level of SHIP1 protein in colonic intestinal tissues of each group. Results: The DAI scores of the mice in the model group were higher than those in the control group, while the DAI scores in the IL-35-treated group were lower than those in the model group (all P < 0.01). Compared with the control group, the colon length was significantly shortened in the model group (P < 0.05), while the colon length of the IL-35-treated group had an increasing trend compared with the model group, but the difference was not statistically significant (P > 0.05). Compared with the model group, microscopic inflammatory infiltration score was decreased and microscopic crypt destruction and score was significantly lower in IL-35-treated group (all P < 0.05). The relative expression of proinflammatory cytokines IL-6, TNF-α and IFN-γ in the colon tissue of IL-35-treated group was decreased compared with the model group, while the relative expression of IL-10 mRNA was higher than that of the model group (all P < 0.05). Compared with the control group, the proportion of M1 macrophages in the model group increased (P < 0.05), while the proportion of M1 macrophages in the IL-35-treated group was lower than that in the model group (P < 0.05). The relative expression of SHIP1 mRNA and protein in the colon tissue of IL-35-treated group was higher than that in the model group (all P < 0.05). Conclusion: IL-35 can inhibit the polarization of M1 macrophages and regulate inflammatory cytokines to promote anti-inflammatory effect on mice with colitis.

Objective: To investigate the effect of Shenmai injection on myocardial cells with oxidative injury and the underlying mechanisms. Methods: Tert-butyl hydroperoxide (t-BHP) was used to induce the oxidative stress in H9c2 myocardial cells. The cell viability and ATP level were evaluated using MTT-colorimetric method and CellTiter-Glo luminescent cell viability assay. The oxygen respiration rate was examined by Clark oxygen electrode. Pyruvate and pyruvate dehydrogenase (PDH) levels were evaluated by ELISA kit. Western blot and quantitative real-time RT-PCR were employed to evaluate the expression of pyruvate dehydrogenase alpha 1(PDHA1) and pyruvate dehydrogenase kinase 1(PDK1). Results: Shenmai injection significantly improved viability and respiration of H9c2 myocardial cells after t-BHP injury (P < 0.05 or P < 0.01). It increased ATP contents by consuming pyruvate and increasing PDH level (P < 0.05 or P < 0.01). Furthermore, Shenmai injection had the tendency to increase protein expression of PDHA1(P < 0.05) and decrease mRNA expression of PDK1 (P>0.05). Conclusion: Shenmai injection protects mitochondria from oxidative stress by increasing PDH level, which indicates that it may improve energy metabolism of myocardial cells.

Objective: To investigate the association of CXCL12 and CXCR4 polymorphisms with the genetic risk and severity of coronary stenosis in patients with coronary artery disease (CAD). Methods: Competitive allele specific PCR(KASP) was performed to identify the genotypes of rs2297630 and rs2322864 polymorphisms in 302 CAD patients and 302 age-and gender-matched healthy controls. The severity of CAD patients was assessed by the Gensini scoring system according to the results of coronary arteriography. The association of rs2297630 and rs2322864 polymorphisms with genetic risk of CAD and Gensini scores were analyzed by unconditional logistic regression and multivariate linear regression respectively. Results: There were significant differences in the genotype and allele frequencies of both rs2297630 and rs2322864 between the CAD group and healthy control (all P < 0.01). Regression analysis showed that rs2297630 polymorphism was associated with genetic risk of CAD and Gensini scores (all P < 0.01). People who carried the AA genotype suffered higher risk of CAD susceptibility and more serious coronary stenosis (all P < 0.01), compared with GG genotype carriers. There was also significant association between rs2322864 polymorphism and genetic risk of CAD (P < 0.01); those who carried the CT genotype had higher risk of CAD (P < 0.01), compared with TT genotype carriers. However, rs2322864 polymorphism was not associated with the severity of coronary stenosis (P>0.05). Conclusions: Gene polymorphism of CXCL12 rs2297630 is associated with the genetic risk of CAD and the severity of coronary stenosis. Moreover, the gene polymorphism of CXCR4 rs2322864 is associated with genetic risk of CAD, but not with the severity of coronary stenosis.

Objective: To establish a cell lines for quality control of prenatal genetic diagnosis. Methods: The recombined SV40LTag-pcDNA3.1(-) vector was constructed and transfected by lipidosome into human amniotic fluid cells with common aneuploidy. Positive clones were screened by G418, and the immortality of transfected cell line was identified. Results: Cell line with karyotype of 46, XY, t(8;19)(q24.3;q13.1) from primary amniotic fluid cells was established. Karyotype analytical results indicated that the cell line at its 15th generation maintained the same karyotype of its primary cell. Conclusion: Gene SV40LT can lead to immortality of amniotic fluid cells, which contributes to preparing cell lines for internal and external quality control in prenatal genetic diagnosis.

In recent years, a large number of studies have achieved tumor targeting by mesenchymal stem cells (MSC)-based delivery system attributed to the tumor tropism of MSCs. Biomacromolecules and antineoplastic drugs loaded on MSC via internalization or cell membrane anchoring can be released or expressed at tumor site to perform their antitumor effects. The genetically modified MSC are extensively studied, however, the applications of MSCs in targeted delivery of antineoplastic drug with small molecules are not well summarized. In this review, MSCs homing mechanism and the distribution of injected MSCs in vivo is introduced; the examples of antitumor drug-primed MSCs and drug loaded MSCs are presented; the drug loading and releasing process from MSCs is also illustrated; finally, challenges and future perspectives of MSCs-based drug delivery system on realizing its full potential are prospected.

Progressive multifocal leukoencephalopathy (PML) is a rare and lethal central nervous demyelinating disease caused by JC polyomavirus (JCV), particularly in patients with impaired immune system. The variation of JCV plays an important role in the pathogenesis of PML, including the recombination of non-coding regulatory region (NCCR), which is closely related to binding sites of transcription factors and affect the level of gene transcription. Nucleotide mutations in VP1 region determine the antigenicity and receptor specificity of JCV, play an important role in cell adsorption, immune-mediation and pathogenicity. In addition, immune cells are also involved in the pathogenesis of PML. T lymphocytes can recognize virus antigens, clear JCV, which are directly related to the prognosis of PML. B lymphocytes can serve as latent sites of JCV, and participate in viral transmission, replication, and coordination of the expression of transcription factors. This paper summarizes the roles of JCV variation and immune cells in pathogenesis of PML.

Promyelocytic leukemia (PML) protein, a tumor suppressor, plays an important role in patients with acute promyelocytic leukemia (APL) receiving arsenic trioxide (As₂O₃) therapy. APL is a M3 subtype of acute myeloid leukemia (AML), which is characterized by expression of PML-RARα (P/R) fusion protein, leading to the oncogenesis. As₂O₃ is currently used as the first-line drug for patients with APL, and the mechanism may be:As₂O₃ directly binds to PML part of P/R protein and induces multimerization of related proteins, which further recruits different functional proteins to reform PML nuclear bodies (PML-NBs), and finally it degraded by SUMOylation and ubiquitination proteasomal pathway. Gene mutations may lead to relapse and drug resistance after As₂O₃ treatment. In this review, we discuss the structure and function of PML proteins; the pathogenesis of APL induced by P/R fusion protein; the involvement of PML protein in treatment of APL patient with As₂O₃; and explain how PML protein mutations could cause resistance to As₂O₃ therapy.

The CCAAT enhancer binding protein α (C/EBP α:p42 and p30), which encoded by CCAAT enhancer binding protein α (C/EBPα) gene, plays a pretty crucial role in the regulation of myeloid hematopoiesis.The disorder of CEBPA gene expression is an pivotal mechanism of acute myeloid leukemia (AML). The result of uncontrolled expression of C/EBP α gene is the over-expression of p30 and the incomplete loss of p42, both of which contribute to the occurrence of AML. Restoring the expression ratio of C/EBP α such as over-expression of p42 or blocking the carcinogenic pathway of p30 seems to be important for the treatment of AML caused by such causes. In order to better guide medical decision-making, this article reviews research progress on C/EBPα in the pathogenesis of AML.

Cancer associated fibroblasts (CAFs) are important components of the tumor microenvironment. Through secreting of multiple growth factors, cytokines and proteases, CAFs play a significant role in regulating the recruitment and function of various innate immune cells and adaptive immune cells in tumor microenvironment. In addition, extracellular matrix secreted by CAFs can also promote the formation of immunosuppression and hypoxia of tumor microenvironment. Here, we review the progress on CAFs in regulation of immune cells and tumor immunity.