Downregulation of Histone H4 Lysine 16 Acetylation Ameliorates Autophagic Flux by Resuming Lysosomal Functions in Ischemic Neurons.

Autophagic/lysosomal dysfunction was a critical pathogenesis of neuronal death after an ischemic stroke, but what drove the impairment of autophagic flux remained elusive. Studies indicated that histone H4 lysine 16 acetylation (H4K16ac) drastically modulated the autophagic/lysosomal signaling pathway. Herein, we investigated whether the autophagic/lysosomal dysfunction in neurons could be restored by altering H4K16ac levels after cerebral ischemia. The rat model of ischemic stroke and the cell ischemia model in HT22 neurons were prepared by middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD), respectively. The result showed that H4K16ac could be effectively reduced by intracerebroventricular administration with MG149 (a H4K16ac inhibitor) after an ischemic stroke. Moreover, attenuated H4K16ac greatly alleviated the autophagic/lysosomal dysfunction in penumbral neurons, as indicated by decreased autophagic substrates of LC3-II, insoluble SQSTM1, and ubiquitinated proteins, accompanied by increased lysosomal cathepsin D. Conversely, treatment with trichostatin A (TSA, a H4K16ac facilitator) aggravated the impairment of autophagic flux. This regulative machinery of H4K16ac on the autophagic/lysosomal signaling pathway was also manifested in the OGD model of HT22 neurons. Furthermore, H4K16ac attenuation-ameliorated autophagic flux significantly alleviated stroke brain injury, as reflected by decreased infarct size, neuron loss, and neurological deficits. Similarly, the H4K16ac inhibition-mitigated autophagic/lysosomal dysfunction markedly promoted neuron survival and cell viability in OGD HT22 neurons. However, H4K16ac downregulation-ameliorated autophagic flux in neurons and thereby induced neuroprotection could be greatly counteracted by the lysosomal inhibitor bafilomycin A1 (Baf-A1). Our data indicate that cerebral ischemia-elevated H4K16ac creates the autophagic/lysosomal dysfunction due to lysosomal inefficiency, suggesting that H4K16ac attenuation benefits poststroke neuroprotection by resuming lysosomal functions in neurons.

Attenuation of histone H4 lysine 16 acetylation (H4K16ac) elicits a neuroprotection against ischemic stroke by alleviating the autophagic/lysosomal dysfunction in neurons at the penumbra.

A modest autophagy benefits neuroprotection while an excessive autophagy leads to neuronal death after cerebral ischemia, but what governs an appropriate autophagy remains to be understood. Studies indicated that acetylation of histone H4 at lysine16 (H4K16ac) strongly modulated autophagic/lysosomal signaling pathway. Thus, this study was to investigate whether the autophagic neuronal injury could be alleviated by amending H4K16ac level after ischemic stroke. A rat model of middle cerebral artery occlusion (MCAO)/reperfusion was prepared to investigate dynamic variations between H4K16ac and autophagy at the penumbra. The results illustrated that the significantly elevated H4K16ac was coupled with dramatically promoted autophagic activity at 4 h after the insult, suggesting H4K16ac tightly controlled autophagic signaling. After that, H4K16ac level was altered by pretreatment with trichostatin A (TSA, a H4K16ac facilitator) and MG149 (a H4K16ac inhibitor), respectively. Four hours after MCAO/reperfusion, the penumbral tissues were obtained to detect the key proteins in autophagic/lysosomal pathway by western blot and immunofluorescence, respectively. Meanwhile, the infarct volume, neurological deficits, and neuron survival were assessed to evaluate the neurological outcomes. The results showed that TSA-promoted H4K16ac led to an excessively up-regulated autophagy resulting in autophagic/lysosomal dysfunction, as indicated by the accumulated autophagic substrates and exacerbated lysosomal inefficiency in neurons. By contrast, MG149-depressed H4K16ac significantly down-regulated autophagic activity and thereby restored the impaired autophagic flux. Consequently, the neurological injury was markedly alleviated in MCAO + MG149 group, compared with that in MCAO group. Our study suggests that the H4K16ac attenuation elicits neuroprotection against ischemic stroke by ameliorating autophagic/lysosomal dysfunction in neurons.

Ginkgo biloba leaf extract (EGb-761) elicits neuroprotection against cerebral ischemia/reperfusion injury by enhancement of autophagy flux in neurons in the penumbra.

OBJECTIVES: Ginkgo biloba leaf extract (EGb-761) injection has been widely used as adjuvant therapy for cerebral stroke in China. However, its underlying pharmacological mechanism is not completely understood. The present study aimed to investigate whether the therapeutic effects of EGb-761 are exerted by modulating autophagy flux. MATERIALS AND METHODS: Ischemic cerebral stroke was prepared in male Sprague-Dawley rats by middle cerebral artery occlusion (MCAO) followed by reperfusion. The MCAO/reperfusion rats were then treated with EGb-761 injection once daily for 7 days. Thereafter, the brain tissues in the ischemic penumbra were obtained to detect the key proteins in the autophagic/lysosomal pathway with Beclin1, LC3, (SQSTM1)/p62, ubiquitin, LAMP-1, cathepsin B, and cathepsin D antibodies by western blot and immunofluorescence. Meanwhile, the infarct volume, neurological deficits, and neuronal apoptosis were assessed to evaluate the therapeutic outcomes. RESULTS: The results illustrated that EGb-761 treatment was not only able to promote the autophagic activities of Beclin1 and LC3-II in neurons, but also could enhance the autophagic clearance, as indicated by reinforced lysosomal activities of LAMP-1, cathepsin B, and cathepsin D, as well as alleviating autophagic accumulation of ubiquitin and insoluble p62 in the MCAO+EGb-761 group, compared with those in the MCAO+saline group. Meanwhile, cerebral ischemia-induced neurological deficits, infarct volume, and neuronal apoptosis were significantly attenuated by 7 days of EGb-761 therapy. CONCLUSION: Our data suggest that EGb-761 injection can elicit a neuroprotective efficacy against MCAO/reperfusion injury, and this neuroprotection may be exerted by enhancement of autophagy flux in neurons in the ischemic penumbra.

Breviscapine confers a neuroprotective efficacy against transient focal cerebral ischemia by attenuating neuronal and astrocytic autophagy in the penumbra.

Breviscapine is a flavonoid derived from a traditional Chinese herb Erigerin breviscapus (Vant.) Hand-Mazz, and has been extensively used in clinical treatment for cerebral stroke in China, but the underlying pharmacological mechanisms are still unclear. In present study, we investigated whether breviscapine could confer a neuroprotection against cerebral ischemia injury by targeting autophagy mechanisms. A cerebral stroke model in Sprague-Dawley rats was prepared by middle cerebral artery occlusion (MCAO), rats were then randomly divided into 5 groups: MCAO+Bre group, rats were treated with breviscapine; MCAO+Tat-Beclin-1 group, animals were administrated with specific autophagy inducer Tat-Beclin-1; MCAO+Bre+Tat-Beclin-1 group, rats were treated with both breviscapine and Tat-Beclin-1, MCAO+saline group, rats received the same volume of physiological saline, and Sham surgery group. The autophagy levels in infarct penumbra were evaluated by western blotting, real-time PCR and immunofluorescence 7days after the insult. Meanwhile, infarct volume, brain water content and neurological deficit score were assessed. The results illustrated that the infarct volume, brain water content and neurofunctional deficiency were significantly reduced by 7days of breviscapine treatment in MCAO+Bre group, compared with those in MCAO+saline group. Meanwhile, the western blotting, quantitative PCR and immunofluorescence showed that the autophagy in both neurons and astrocytes at the penumbra were markedly attenuated by breviscapine admininstration. Moreover, these pharmacological effects of breviscapine could be counteracted by autophagy inducer Tat-Beclin-1. Our study suggests that breviscapine can provide a neuroprotection against transient focal cerebral ischemia, and this biological function is associated with attenuating autophagy in both neurons and astrocytes.

Puerarin provides a neuroprotection against transient cerebral ischemia by attenuating autophagy at the ischemic penumbra in neurons but not in astrocytes.

Puerarin is an isoflavone derived from the Chinese medical herb of Radix puerariae (kudzu root), and has been widely used in the treatment for ischemic stroke in China. However, its underlying pharmacological mechanisms are still not understood. This study was to investigate the efficacy of puerarin on autophagy in the ischemic penumbra after cerebral stroke. A model of cerebral stroke in Sprague-Dawley rats was prepared by middle cerebral artery occlusion (MCAO); rats were then randomly divided into 5 groups: MCAO+Pue group (rats were treated with puerarin), MCAO+Pue+Tat-Beclin-1 group (rats were administrated with both puerarin and autophagy inducer Tat-Beclin-1), MCAO+Tat-Beclin-1 group (rats were treated with Tat-Beclin-1), MCAO+saline group (rats were administrated with the same volume of physiological saline), and sham surgery group. The autophagy levels in infarct penumbra were evaluated by western blotting, real-time PCR and immunofluorescence 14days after the insult. Meanwhile, the neurological deficit score, brain water content and infarct volume were assessed. The results illustrated that the cerebral infarct volume, cerebral edema and neurological deficiency were significantly alleviated by puerarin treatment. Western blotting and the quantitative PCR revealed that the autophagy level in the penumbra was markedly reduced by puerarin administration. However, these effects of puerarin could be counteracted by Tat-Beclin-1. Additionally, double immunofluorescence showed that neuronal autophagy was markedly attenuated by puerarin treatment, whereas astrocytic autophagy was only mildly reduced. Our study suggests that puerarin could confer a neuroprotection against cerebral ischemia, and this biological function is associated with attenuating autophagy in neurons but not in astrocytes.

Latency-associated protein Rv2660c of Mycobacterium tuberculosis augments expression of proinflammatory cytokines in human macrophages by interacting with TLR2.

BACKGROUND: One-third of the world's human population is latently infected with Mycobacterium tuberculosis (Mtb), and individuals with latent infection have a 10% risk of developing active tuberculosis (TB) disease in their lifetime. Rv2660c (encoding a hypothetical protein) is closely correlated with dormancy of Mtb. Studies have found that Rv2660c was preferentially expressed during latent infection for adaptation to lack of nutrition and hypoxia; however, the precise function is unknown. Identification and characterization of Rv2660c is crucial to understand host-pathogen interactions and to develop drug targets and vaccine candidates. METHODS: This study investigated the functional roles and the related signaling mechanism of Rv2660c interacting with human macrophages. RESULTS: The results showed that either rBCG-Rv2660c strain (expressing Rv2660c protein) or recombinant purified Rv660c protein was able to stimulate peripheral blood mononuclear cells (PBMCs) and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells to secrete the important cytokines interleukin (IL)-1ß, IL-8, tumor necrosis factor (TNF)-α, and IL-12p70, which are essential for granuloma formation and maintenance. Meanwhile, Rv2660c recognized Toll-like receptor 2 (TLR2) to activate macrophages. CONCLUSION: The results suggested that Rv2660c was able to stimulate human macrophages to provoke the secretion of proinflammatory cytokines by interacting with TLR2 signaling, and the proinflammatory responses elicited by Rv2660c might be an important way to maintain latency of Mtb.

Association between IFN-γexpression and pathological damage of lung tissue during pulmonary tuberculosis / 实用医学杂志

Objective To investigate the relationship between the expression of IFN-γ, IL-2 and TNF-αand pathological damage of lung tissue, and to provide reference values for protection and treatment of pulmonary tuberculosis. Methods BALB/c mice models of pulmonary tuberculosis were established by infection with Mycobacterium tuberculosis H37Rv through the caudal vein. At 0 (not infected),2,4,6W and 8W after infection, the bacterial loads in the lung were performed and the pathological damage of the lung was evaluated, respectively. At the same time, the expression of IFN-γ,IL-2 and TNF-α-positive cells in the lung tissues was detected by immunohistochemistry. Results Two weeks after infection, the expression of IFN-γ, IL-2 and TNF-αpositive cells was obviously increased compared with those at 0 week. The proportion of IFN-γ-positive cells were obviously decreased at 6 weeks (9.25%) to 8 weeks (5.67%) after infection with Mycobacterium tuberculosis H37Rv. At the same stage, there is a limited decrease tendency of TNF-α and IL-2 expression compared with those at 4 weeks after infection, and there was no significant difference compared with those at 2W or 4W after infection. At 8 weeks after infection, the bacterial loads (8.43) in the lung and the scores(17) of pathological damage were significantly increased compared with those at 6 weeks after infection. Conclusion The high expression of IFN-γ, IL-2 and TNF-αprotect lung tissues against MTB infection at the early stage. The decrease of IFN-γexpression is responsible for serious damage of the lung tissues in the persistent infection.

Study on immunogenicity elicited by a recombinant vaccine of rBCG-Rv3133c to fight against dormancy Mycobacterium tuberculosis / 生物医学工程学杂志

To obtain a vaccine to defend from dormancy Mycobacterium tuberculosis, we constructed the recombinant Bacilli Calmette-Guérin (BCG) vaccine with Rv3133c encoding dormancy-correlated transcriptional regulatory protein DosR in Mycobacterium tuberculosis as a target gene, and evaluated its immunogenicity in BALB/c mice. In this study, we constructed the recombinant plasmids of rpMV361-Rv3133c using gene colon technology. We then transformed BCG strains with above-mentioned plasmids to obtain recombinant vaccine of rBCG-Rv3133c. We used the rBCG strains successfully constructed to vaccinate in BALB/c mice. 30d and 180d after immunization, the specific antibody titers were determined to investigate humoral responses induced by recombinant vaccine. We detected changes of splenocyte subsets of CD4+T, CD8+ T cells and cytokine of IFN-gamma secreted by splenocytes for evaluation of cellular immune responses. The results showed that the rBCG-Rv3133c was able to induce higher levels of antibody titer, stronger proliferative responses and higher IFN-gamma production comparing with BCG vaccine. The results also suggested that this recombinant vaccine was a more efficacious tuberculosis vaccine for further study.

Differentiation of rat bone marrow stromal stem cells into neuron-like cells induced by breviscapine / 解剖学报

Objective To explore the feasibility of differentiation into neural cells from bone marrow stromal cells(BMSCs) in vitro by breviscapine,and to offer reference for the applying of BMSCs. Methods The bone marrow stromal cells of SD rat were separated and purified by passsage culture.After phenotype characterization,the 4th passage of BMSCs was exposed to breviscapine. Differentiation was observed under phase contrast microscope every 6 hours and the cells were stained immunocytochemically with neuronspecific enolase(NSE)and glial fibrillary acidic protein(GFAP).The induced cells activity was detected by MTT. NSE and GFAP were determined by flow cytometry and RT-PCR. Results The BMSCs were CD44~+,CD54~+,CD34~-. After 18 hours of induction, cytoplasm of BMSCs partly contracted with protruding;The immunocytochemical staining was performed on cells after induction for 24 hours,the rates of NSE and GFAP staining positive were(48.7±3.4)% and(56.8±4.2)%.By using flow cytometer, expressions of NSE and GFAP showed much higher in induced group than that in control group. By using RT-PCR, expressions of NSE and GFAP were positive in induced group. Conclusion Breviscapine could induce adult rat BMSCs to differentiate into neuron and glial cells.

Repair of sciatic nerve defects with tissue engineered nerves constructed with marrow stromal cells / 中国组织工程研究

BACKGROUND: Schwann calls are seed cells for constructing tissue engineered peripheral nerves. But their in vitro isolation, culture and purification are difficult. Acellular nerve allografts (ANA) have a great effect on repairing peripheral nerve defects, and it can induce marrow stromal cells (MSCs) into Schwann-like calls. Accordingly, MSCs can be used as seed calls theoretically in constructing tissue engineered peripheral nerves, instead of Schwann cells OBJECTIVE: To observe the repairing effect of tissue engineered peripheral nerves constructed with MSCs on sciatic nerve defects and to assess the feasibility of repairing peripheral nerve defects with MSCs as seed calls. DESIGN, TIME AND SE'I-rlNG: A randomized control animal experiment was conducted in the laboratory of Basic Medicine Collage of Dali Collage from July to December in 2008.MATERIALS: A total of 30 SD rats were divided randomly into 3 groups, with 10 in each group. In MSCs+ ANA group, end-to-end anastomosis were performed with 10/0 non traumatic suture to broken ends of rat sciatic nerves and tissue engineered nerves cultured using MSCs combined with ANA; In ANA group, ANA were bridged to broken ends of sciatic nerves; In METHODS: The 10ram sciatic nerve defects in rats were repaired using MSCs-constructed tissue engineered peripheral nerves, whose repairing effects were evaluated at week 12 post transplant through sciatic function index (SFI), gastrocnemius wet weight recovery rate, S-100 immunohistochemical staining and electron microscope observation, etc. MAIN OUTCOME MEASURES: Morphological changes of calls were observed during the culture of compounds; SFI and gastrocnemius wet weight recovery rates were detected after transplantation; New myelinization, axon growth and nerve fiber distdbuUon were observed with toluidine blue staining; Schwann calls growth and nerve fiber regeneration were observed with transmission electron microscope combined with S-100 protein immunohistochemical staining method.RESULTS: The detection results showed that SFI and gastrocnemius wet weight recovery rate were higher in the MSCs+ ANA group than in the ANA group (P < 0.05). Compared the ANA group, the MSCs+ ANA group had more S-100 proteins expressed in its compounds obviously, and the number, the diameter of its myelinated nerve fibers as well as its myelin sheath thickness were all greater than those of the ANA group (P < 0.05). The repairing effect of the MSCs+ ANA group was close to that of the autotransplantation group.CONCLUSION: MSCs-constructed tissue engineered nerves have a better effect on repairing peripheral nerve defects than ANA, and MSCs as seed cells have a high value in peripheral nerve tissue engineering.

Puerarin reduces ethanol-induced-apoptosis of spermatogenic cells in rat testis / 基础医学与临床

Objective To investigate potential protection by protect against ethanol-induced apoptosis of spermato-genie cells in rat testis. Methods Thirty SD adult male rats were randomly divided into three groups: normal group, alcohol group and puerarin group. At 40th day, BCL-2 and BAX of spermatogenic cells of testis tissue were checked by RT-PCR and immunohistochemistry; Apoptosis of spermatogenic cells was determined by TUNEL. Re-suits The results of RT-PCR and immunohistochemistry indicated that BCL-2 and BAX of spermatogenie cells were not significanty different between puerarin group and normal group, but there was the significant difference between alcohol group and puerarin group (P <0.01). Apoptosis of spermatogenic cells in alcohol group was significantly higher than normal group. Conclusion Spermatogenic cells could generate apoptosis by changing the expression of BCL-2 and BAX. Puerarin could inhibit this damage of didymus by alcohol.

Puerarin reduces ethanol-induced-apoptosis of spermatogenic cells in rat testis / 基础医学与临床

Objective To investigate potential protection by protect against ethanol-induced apoptosis of spermatogenic cells in rat testis.Methods Thirty SD adult male rats were randomly divided into three groups: normal group,alcohol group and puerarin group.At 40th day,BCL-2 and BAX of spermatogenic cells of testis tissue were checked by RT-PCR and immunohistochemistry;Apoptosis of spermatogenic cells was determined by TUNEL.Results The results of RT-PCR and immunohistochemistry indicated that BCL-2 and BAX of spermatogenic cells were not significanty different between puerarin group and normal group,but there was the significant difference between alcohol group and puerarin group(P

Anatomic background of chronic spontaneous pain of neural entrapment syndrome in the inguinal region / 中国组织工程研究

BACKGROUND: The main clinical manifestation of the nerve entrapment syndrome in the inguinal region is chronic and spontaneous pain of the scrotal region and proximal ventro-medial thigh region. Few reports have discussed the anatomic background of this kind of pain with special reference to skin innervation.OBJECTIVE: To study the features of clinical anatomy in entrapment of nerve for providing anatomic basis for preventing and treating entrapment of nerves in the inguinal region.DESIGN: Observational study based on cadavers.SETTING: Anatomical department in a university.MATERIALS: Fifty halves of twenty-five adult male cadavers that were routinely embalmed and fixed by the Anatomical Department of Dali University from January 1998 to December 2000.METHODS: Cutaneous nerves in the inguinal region in 50 halves of 25adult male cadavers were observed, measured and drawn.tionship of the genital branch of the genitofemoral nerve to the inguinal canal.RESULTS: In addition to cutaneous branches originating from the iliohypogastric nerve in 3 of 50 cases(6% ), cutaneous branches from the ilioinguinal nerve were found in the inguinal region in 45 of 50 halves(90% ),cutaneous nerves from the genital branch of genitofemoral nerve were in 21 of 50 halves(42% ), the unions of the ilioinguinal nerve and genital branch of the genitofemoral nerve were in 6 of 50 sides(12% ), and branches from the femoral branch of the genitofemoral nerve were in 4 of 50 sides(8% ) . The genital branch of genitofemoral nerve and the ilioinguinal nerve united at three the canal(1 case). The cutaneous branches of the genital branch were found to perforating the transversus abdominis and the obliquus internus abdominis via the border between the ligament and the aponeurosis of obliquus externus abring after being united with the ilioinguinal nerve.CONCLUSION: The courses of cutaneous nerves in the inguinal region vary considerably, and the anatomic variations of these nerves may be a principal cause for nerve entrapment.