Neuroprotective potential of traditionally used medicinal plants of Manipur against rotenone-induced neurotoxicity in SH-SY5Y neuroblastoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Alternanthera sessilis (L.) R. Br. ex DC., Eryngium foetidum L., and Stephania japonica (Thunb.) Miers plants are traditionally used to treat various central nervous system disorders like paralysis, epilepsy, seizure, convulsion, chronic pain, headache, sleep disturbances, sprain, and mental disorders. However, their possible neuroprotective effects have not been evaluated experimentally so far. AIM OF THE STUDY: The study aims to examine the neuroprotective potential of the three plants against cytotoxicity induced by rotenone in SH-SY5Y neuroblastoma cells and assess its plausible mechanisms of neuroprotection. MATERIALS AND METHODS: The antioxidant properties of the plant extracts were determined chemically by DPPH and ABTS assay methods. The cytotoxicity of rotenone and the cytoprotective activities of the extracts were evaluated using MTT assays. Microtubule-associated protein 2 (MAP2) expression studies in cells were performed to assess neuronal survival after rotenone and extract treatments. Mitochondrial membrane potential and intracellular levels of reactive oxygen species were evaluated using Rhodamine 123 and DCF-DA dye, respectively. Catalase, glutathione peroxidase, and superoxide dismutase activities were also measured. Apoptotic nuclei were examined using DAPI staining. Liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS) analysis of the plant extracts was also performed. RESULTS: The methanol extracts of A. sessilis, S. japonica, and E. foetidum showed excellent free radical scavenging activities. MAP2 expression studies show that A. sessilis and S. japonica have higher neuroprotective effects against rotenone-induced neurotoxicity in SH-SY5Y cells than E. foetidum. Pre-treating cells with the plant extracts reverses the rotenone-induced increase in intracellular ROS. The plant extracts could also restore the reduced mitochondrial membrane potential induced by rotenone treatment and reinstate rotenone-induced increases in catalase, glutathione peroxidase, and superoxide dismutase activities. All the extracts inhibited rotenone-induced changes in nuclear morphology and DNA condensation, an early event of cellular apoptosis. LC-QTOF-MS analysis of the plant extracts shows the presence of neuroprotective compounds. CONCLUSIONS: The plant extracts showed neuroprotective activities against rotenone-treated SH-SY5Y cells through antioxidant and anti-apoptotic mechanisms. These findings support the ethnopharmacological uses of these plants in treating neurological disorders. They probably are a good source of neuroprotective compounds that could be further explored to develop treatment strategies for neurodegenerative diseases like Parkinson's disease.

JPT2 Affects Trophoblast Functions and Macrophage Polarization and Metabolism, and Acts as a Potential Therapeutic Target for Recurrent Spontaneous Abortion.

Recurrent spontaneous abortion (RSA) is a pregnancy-related condition with complex etiology. Trophoblast dysfunction and abnormal macrophage polarization and metabolism are associated with RSA; however, the underlying mechanisms remain unknown. Jupiter microtubule-associated homolog 2 (JPT2) is essential for calcium mobilization; however, its role in RSA remains unclear. In this study, it is found that the expression levels of JPT2, a nicotinic acid adenine dinucleotide phosphate-binding protein, are decreased in the villous tissues of patients with RSA and placental tissues of miscarried mice. Mechanistically, it is unexpectedly found that abnormal JPT2 expression regulates trophoblast function and thus involvement in RSA via c-Jun N-terminal kinase (JNK) signaling, but not via calcium mobilization. Specifically, on the one hand, JPT2 deficiency inhibits trophoblast adhesion, migration, and invasion by inhibiting the JNK/atypical chemokine receptor 3 axis. On the other hand, trophoblast JPT2 deficiency contributes to M1 macrophage polarization by promoting the accumulation of citrate and reactive oxygen species via inhibition of the JNK/interleukin-6 axis. Self-complementary adeno-associated virus 9-JPT2 treatment alleviates embryonic resorption in abortion-prone mice. In summary, this study reveals that JPT2 mediates the remodeling of the immune microenvironment at the maternal-fetal interface, suggesting its potential as a therapeutic target for RSA.

[Effect and mechanism of Danggui Buxue Decoction-containing serum in mitigating H9c2 cell injury caused by exposure to intermittent low oxygen].

This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.

Microtubule-associated proteins WDL5 and WDL6 play a critical role in pollen tube growth in Arabidopsis thaliana.

Morphological response of cells to environment involves concerted rearrangements of microtubules and actin microfilaments. A mutant of WAVE-DAMPENED2-LIKE5 (WDL5), which encodes an ethylene-regulated microtubule-associated protein belonging to the WVD2/WDL family in Arabidopsis thaliana, shows attenuation in the temporal root growth reduction in response to mechanical stress. We found that a T-DNA knockout of WDL6, the closest homolog of WDL5, oppositely shows an enhancement of the response. To know the functional relationship between WDL5 and WDL6, we attempted to generate the double mutant by crosses but failed in isolation. Close examination of gametophytes in plants that are homozygous for one and heterozygous for the other revealed that these plants produce pollen grains with a reduced rate of germination and tube growth. Reciprocal cross experiments of these plants with the wild type confirmed that the double mutation is not inherited paternally. These results suggest a critical and cooperative function of WDL5 and WDL6 in pollen tube growth.

Swimming and L-arginine loaded chitosan nanoparticles ameliorates aging-induced neuron atrophy, autophagy marker LC3, GABA and BDNF-TrkB pathway in the spinal cord of rats.

Aging is associated with muscle atrophy, and erosion and destruction of neuronal pathways in the spinal cord. The study aim was to assess the effect of swimming training (Sw) and L-arginine loaded chitosan nanoparticles (LA-CNPs) on the sensory and motor neuron population, autophagy marker LC3, total oxidant status/total antioxidant capacity, behavioural test, GABA and BDNF-TrkB pathway in the spinal cord of aging rats. The rats were randomized to five groups: young (8-weeks) control (n = 7), old control (n = 7), old Sw (n = 7), old LA-CNPs (n = 7) and old Sw + LA-CNPs (n = 7). Groups under LA-CNPs supplementation received 500 mg/kg/day. Sw groups performed a swimming exercise programme 5 days per week for 6 weeks. Upon the completion of the interventions the rats were euthanized and the spinal cord was fixed and frozen for histological assessment, IHC, and gene expression analysis. The old group had more atrophy in the spinal cord with higher changes in LC3 as an indicator of autophagy in the spinal cord compared to the young group (p < 0.0001). The old Sw + LA-CNPs group increased (improved) spinal cord GABA (p = 0.0187), BDNF (p = 0.0003), TrkB (p < 0.0001) gene expression, decreased autophagy marker LC3 protein (p < 0.0001), nerve atrophy and jumping/licking latency (p < 0.0001), improved sciatic functional index score and total oxidant status/total antioxidant capacity compared to the old group (p < 0.0001). In conclusion, swimming and LA-CNPs seems to ameliorate aging-induced neuron atrophy, autophagy marker LC3, oxidant-antioxidant status, functional restoration, GABA and BDNF-TrkB pathway in the spinal cord of aging rats. Our study provides experimental evidence for a possible positive role of swimming and L-arginine loaded chitosan nanoparticles to decrease complications of aging.

[Zexie Decoction regulates Akt/TFEB signaling pathway to promote lipophagy in hepatocytes].

Taking lipophagy as the breakthrough point, we explored the mechanism of Zexie Decoction(ZXD) in improving lipid metabolism in the hepatocyte model induced by palmitic acid(PA) and in the animal model induced by high-fat diet(HFD) on the basis of protein kinase B(Akt)/transcription factor EB(TFEB) signaling pathway. Co-localization was carried out for the microtubule-associated protein light chain 3(LC3) plasmid labeled with green fluorescent protein(GFP) and lipid droplets(LDs), and immunofluorescence co-localization for liver LC3 of HFD mice and perilipin 2(PLIN2). The results showed that ZXD up-regulated the expression of LC3, reduced lipid accumulation in hepatocytes, and increased the co-localization of LC3 and LDs, thereby activating lipo-phagy. Western blot results confirmed that ZXD increased autophagy-related protein LC3Ⅱ/LC3Ⅰ transformation ratio and lysosome-associated membrane protein 2(LAMP2) in vivo and in vitro and promoted the degradation of sequestosome-1(SQSTM1/p62)(P<0.05). The results above jointly explained that ZXD regulated lipophagy. Furthermore, ZXD activated TFEB expression(P<0.05) and reversed the PA-and HFD-induced decrease of TFEB nuclear localization in hepatocytes(P<0.05). Meanwhile, ZXD activated liver TFEB to up-regulate the expression of the targets Lamp2, Lc3 B, Bcl2, and Atg5(P<0.05). Additionally, ZXD down-regulated the protein level of p-Akt upstream of TFEB in vivo and in vitro. In conclusion, ZXD may promote lipophagy by regulating the Akt/TFEB pathway.

Danshen injection induces autophagy in podocytes to alleviate nephrotic syndrome via the PI3K/AKT/mTOR pathway.

BACKGROUND: Danshen injection (DSI) is an agent extracted from the Salvia miltiorrhiza Bunge, a natural drug commonly used to alleviate kidney diseases. However, the material basis and therapeutic effects of DSI on nephrotic syndrome (NS) remain unclear. PURPOSE: To investigate the material basis of DSI and the therapeutic effects and underlying mechanisms of NS. METHODS: NS models were established using adriamycin-induced BALB/c mice and lipopolysaccharide-induced mouse podocytes (MPC-5). Following DSI and prednisone administration, kidney coefficients, 24 h urine protein, blood urea nitrogen, and serum creatinine levels were tested. Histomorphology was observed by periodic acid-Schiff staining and hematoxylin and eosin staining of the kidney sections. The glomerular basement membrane and autophagosomes of the kidneys were observed using transmission electron microscopy. Nephrin and desmin levels in the glomeruli were tested using immunohistochemistry. The viability of MPC-5 cells was tested using cell counting kit-8 after chloroquine and rapamycin administration in combination with DSI. The in vivo and in vitro protein levels of phosphatidylinositol 3-kinase (PI3K), AKT, phosphorylated AKT (Ser473), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), beclin1, cleaved caspase-3, and caspase-3 were detected using western blotting. RESULTS: Our results showed that DSI contained nine main components: caffeic acid, danshensu, lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, and 3, 4-Dihydroxybenzaldehyde. In in vivo studies, the NS mice showed renal function and pathological impairment. Podocytes were damaged, with decreased levels of autophagy and apoptosis, accompanied by inhibition of the PI3K/AKT/mTOR signaling. DSI administration resulted in improved renal function and pathology in NS mice, with the activation of autophagy and PI3K/AKT/mTOR signaling in the kidneys. Additionally, podocytes were less damaged and intracellular autophagosomes were markedly increased. In vitro studies have shown that DSI activated MPC-5 autophagy and reduced apoptosis via the PI3K/AKT/mTOR pathway. CONCLUSION: Collectively, this study demonstrated that DSI activated podocyte autophagy and reduced apoptosis via the PI3K/AKT/mTOR signaling, ultimately attenuating NS. Our study clarified the main components of DSI and elucidated its therapeutic effects and potential mechanisms for NS, providing new targets and agents for the clinical treatment of NS.

[Effect of heat-reinforcing needling on expression of serum inflammatory factors and autophagy of knee joint synovial tissue in rheumatoid arthritis rabbits with cold syndrome].

OBJECTIVE: To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis (RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA. METHODS: Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups (n=10 rabbits in each group). The model of RA with cold syndrome was established by Freund's adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at "Zusanli" (ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor (TNF) -α, interleukin (IL)-1ß, IL-6 and C-creactive protein (CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1), microtubule-associated protein light chain 3B (LC3B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3 (NLRP3) and nuclear factor-κB (NF-κB). RESULTS: Compared with the normal group, the circumference of the knee joint was increased (P<0.01), the skin temperature was decreased (P<0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1ß, IL-6, and CRP were increased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3BⅡ/LC3BⅠof synovial tissue were significantly decreased (P<0.01), the mRNA expressions of NLRP3 and NF-κB were increased (P<0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased (P<0.01), whlie the skin temperature was increased (P<0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1ß, IL-6 and CRP were decreased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3B Ⅱ/LC3B Ⅰ were increased (P<0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased (P<0.01) in the heat-reinforcing needling and agonist groups. CONCLUSION: Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1ß, IL-6 and CRP.

Ginseng volatile oil prolongs the lifespan and healthspan of Caenorhabditis elegans.

Ginseng volatile oil (GVO) is one of the main components of ginseng and has antibacterial and anti-inflammatory properties. In this study, gas chromatography-mass spectrometry (GC-MS) was applied to characterize GVO chemical composition, and 73 volatile components were detected from GVO. Caenorhabditis elegans was used as animal model to further elucidate the antioxidant and anti-aging effects of GVO in vivo. The results suggested that GVO significantly prolonged the lifespan of C. elegans and promoted its health without damaging its reproductive capacity. In addition, GVO increased the antioxidant capacity and survival rate of nematodes after heat shock. Transcriptional sequencing showed that autophagy-related genes atg-4.2, atg-7, lgg-2, and cyd-1 were up-regulated, and superoxide dismutase 1 (sod-1) expression was increased after GVO pretreatment. Considering the role of autophagy and antioxidant in aging, the expression of autophagy substrate P62 protein in BC12921 strain was analyzed and found to decrease by more than 50.00% after treatment with GVO. In addition, the lifespan of SOD-1 mutant nematodes was not significantly different from that of the control group. SOD activity and autophagy were activated, which is a clear expression of hormesis. All these results suggest that GVO prolongs the lifespan and healthspan of C. elegans, and its biological functions may be related to hormesis.

[Effect of mild moxibustion on pain and expression of LC3 and Bax in spinal cord in cervical spondylotic radiculopathy rats].

OBJECTIVE: To observe the effect of mild moxibustion (Moxi) at "Dazhui" (GV14) on neuropathic pain, expression of autophagy and apoptosis factor LC3 and Bax proteins and mRNAs in the spinal cord tissue in rats with cervical spondylotic radiculopathy (CSR), so as to explore its underlying mechanism underlying relief of CSR-induced pain. METHODS: Forty rats (half male half female) were randomly divided into blank control, model, Moxi, Moxi+autophagy inhibitor 3-methyladenine (3-MA, Moxi+3-MA) groups, with 10 rats in each group. The CSR model was established by loose ligature of the local cervical nerve roots. Three days after modeling, mild Moxi was applied to GV14 for 10 min, once daily for 7 days. Rats of the Moxi+3-MA group received intraperitoneal injection of 3-MA(1 mL, 15 mg/kg+ saline) before Moxi, once daily for 7 consecutive days. Rats of the model and Moxi groups were also given normal saline (i.p., 1 mL), once daily for 7 days. The gait behavior score (1-3 points) was scaled according to the rats' pain reaction and foot paw contracture produced walking disorder and the mechanical pain threshold (MPT) was detected before and after the treatment. The expression of spinal cord LC3 and Bax proteins and mRNAs were detected by immunohistochemistry and quantitative RT-PCR, respectively. RESULTS: Compared with the blank control group, the gait disorder score, and percentage of Bax positive cells and expression of Bax mRNA were significantly increased (P<0.01, P<0.05), and MPT was markedly decreased in the model group (P<0.01). After the treatment, the gait disorder score, percentage of Bax positive cells and Bax mRNA expression were significantly down-regulated (P<0.01, P<0.05), while the MPT and percentage of LC3 positive cells and LC3 mRNA expression were considerably increased (P<0.01, P<0.05) in both Moxi and Moxi+3-MA groups. The therapeutic effects of mild Moxi were remarkably superior to those of Moxi+3-MA in downregulating gait disorder score, Bax positive cell percentage and Bax mRNA expression, and in up-regulating MPT, LC3 positive cell percentage and LC3 mRNA expression (P<0.05), suggesting a reduction of the function of mild Moxi after administration of 3-MA. CONCLUSION: Mild Moxi at GV14 can relieve neuropathic pain in CSR rats, which may be related to its functions in up-regulating LC3 autophagy, thereby inhibiting the expression of Bax pro-apoptotic protein in spinal cord to reduce apoptosis and to repair nerve injury.

Knock-down of gene expression throughout meiosis and pollen formation by virus-induced gene silencing in Arabidopsis thaliana.

Through the inactivation of genes that act during meiosis it is possible to direct the genetic make-up of plants in subsequent generations and optimize breeding schemes. Offspring may show higher recombination of parental alleles resulting from elevated crossover (CO) incidence, or by omission of meiotic divisions, offspring may become polyploid. However, stable mutations in genes essential for recombination, or for either one of the two meiotic divisions, can have pleiotropic effects on plant morphology and line stability, for instance by causing lower fertility. Therefore, it is often favorable to temporarily change gene expression during meiosis rather than relying on stable null mutants. It was previously shown that virus-induced gene silencing (VIGS) can be used to transiently reduce CO frequencies. We asked if VIGS could also be used to modify other processes throughout meiosis and during pollen formation in Arabidopsis thaliana. Here, we show that VIGS-mediated knock-down of FIGL1, RECQ4A/B, OSD1 and QRT2 can induce (i) an increase in chiasma numbers, (ii) unreduced gametes and (iii) pollen tetrads. We further show that VIGS can target both sexes and different genetic backgrounds and can simultaneously silence different gene copies. The successful knock-down of these genes in A. thaliana suggests that VIGS can be exploited to manipulate any process during or shortly after meiosis. Hence, the transient induction of changes in inheritance patterns can be used as a powerful tool for applied research and biotechnological applications.

[Effect of transcutaneous auricular vagus nerve stimulation on the expressions of GFAP and MAP2 in ischemic penumbra of rats with middle cerebral artery ischemia].

OBJECTIVE: To observe the effects of transcutaneous auricular vagus nerve stimulation (taVNS) on the motor function and the expression of glial fibrillary acidic protein (GFAP) and microtubule associated protein 2 (MAP2) in cerebral ischemic penumbra of rats with middle cerebral artery occlusion (MCAO) and explore the mechanism of taVNS in the improvement of motor function in MCAO rats. METHODS: A total of 48 male SD rats were randomized into a sham-operation group, a model group, a transcutaneous auricular non-vagus nerve stimulation (tnVNS) group and a taVNS group, with 12 rats in each group. The suture-occluded method was adopted to prepare MCAO rat model. The auricular rim was stimulated in the tnVNS group and the concha stimulated in the taVNS group, 2 mA in intensity, 10 Hz in frequency, 30 min each time, once a day, for 14 days consecutively. The nerve functional assessment was recorded in each group. The expressions of nicotinic acetylcholine receptor (α7nAchR) in the cerebral ischemic penumbra and the spleen were detected by using Western blot. With the immunofluorescence, the expressions of GFAP and MAP2 were detected. RESULTS: After modeling, compared with the sham-operation group, the nerve functional score was increased in the model group, the tnVNS group and the taVNS group (P<0.01), suggesting the success of modeling. After treatment, the score was increased in the model group (P<0.01) as compared with the sham-operation group. Compared with the model group, the neurological deficit score was reduced in the taVNS group (P<0.01). Compared with the sham-operation group, GFAP expression was increased and MAP2 expression was reduced remarkably in the cerebral ischemic penumbra in the model group (P<0.05). In comparison with the model group, GFAP expression was reduced, while MAP2 expression was increased remarkably in the cerebral ischemic penumbra in the taVNS group (P<0.05). There were no significant differences in the abovementioned indexes between the model group and tnVNS group (P>0.05). The differences in the expression of α7nAchR in the cerebral ischemic penumbra and the spleen had no statistical significance among groups (P>0.05). CONCLUSION: TaVNS is effective on neuroprotection in MCAO rats, which may be related to its function of inhibition of GFAP expression and promotion of MAP2 expression in the ischemic penumbra.

Salvianolic acid A relieves cognitive disorder after chronic cerebral ischemia: Involvement of Drd2/Cryab/NF-κB pathway.

Chronic cerebral ischemia (CCI) refers to long-term hypoperfusion of cerebral blood flow with the main clinical manifestations of progressive cognitive impairment. The pathological mechanism of CCI is complex, and there is a lack of effective treatments. Salvianolic acid A (SalA) is a neuroprotective extract of Salvia miltiorrhiza with the effects of anti-inflammation and anti-apoptosis. In this study, the effect of SalA on cognitive function and Drd2/Cryab/NF-κB signaling pathway in rats with CCI was investigated. Morris water maze and open field test were used to observe the effects of SalA on the cognitive function of CCI rats. The pathological changes in the brain were observed by HE, Nissl, and LFB staining. TUNEL staining, enzyme-linked immunosorbent assay, and western blot analysis were used to detect the inflammatory and apoptosis in the cortex and hippocampus. The expression of Drd2/Cryab/NF-κB pathway-related molecules and Drd2 localization were detected by western blotting and dual immunofluorescence, respectively. SH-SY5Y cells were exposed to chronic hypoglycemic and hypoxic injury in vitro, and Drd2 inhibitor haloperidol was used to verify the involved pathway. The results showed that SalA could improve the cognitive function of CCI rats, reduce pathological damage of cortex and hippocampus, inhibit neuroinflammation and apoptosis, and suppress the activation of NF-κB by regulating Drd2/Cryab pathway. And SalA inhibited NF-κB activation and nuclear translocation in SH-SY5Y cells by upregulating Drd2/Cryab pathway, which was reversed by haloperidol interference. In conclusion, SalA could relieve CCI-induced cognitive impairment in rats, at least partly through the Drd2/Cryab/NF-κB pathway.

Tissue-Specific Gamma-Flicker Light Noninvasively Ameliorates Retinal Aging.

Aging is a risk factor for multiple retinal degeneration diseases. Entraining brain gamma oscillations with gamma-flicker light (γFL) has been confirmed to coordinate pathological changes in several Alzheimer's disease mouse models and aged mice. However, the direct effect of γFL on retinal aging remains unknown. We assessed retinal senescence-associated beta-galactosidase (ß-gal) and autofluorescence in 20-month-old mice and found reduced ß-gal-positive cells in the inner retina and diminished lipofuscin accumulation around retinal vessels after 6 days of γFL. In immunofluorescence, γFL was further demonstrated to ameliorate aging-related retinal changes, including a decline in microtubule-associated protein 1 light chain 3 beta expression, an increase in complement C3 activity, and an imbalance between the anti-oxidant factor catalase and pro-oxidant factor carboxymethyl lysine. Moreover, we found that γFL can increase the expression of activating transcription factor 4 (ATF4) in the inner retina, while revealing a decrease of ATF4 expression in the inner retina and positive expression in the outer segment of photoreceptor and RPE layer for aged mice. Western blotting was then used to confirm the immunofluorescence results. After mRNA sequencing (NCBI Sequence Read Archive database: PRJNA748184), we found several main mechanistic clues, including mitochondrial function and chaperone-mediated protein folding. Furthermore, we extended γFL to aged Apoe-/- mice and showed that 1-m γFL treatment even improved the structures of retinal-pigment-epithelium basal infolding and Bruch's membrane. Overall, γFL can orchestrate various pathological characteristics of retinal aging in mice and might be a noninvasive, convenient, and tissue-specific therapeutic strategy for retinal aging.

[Effect of acupuncture serum on expression of microtubule associated protein-2 and nerve growth associated protein-43 in Mg2+-free-cultured hippocampal neurons of neonatal rats].

OBJECTIVE: To observe the effect of acupuncture serum on the expression of microtubule associated protein-2 (MAP-2) and nerve growth associated protein-43 (GAP-43) in cultured hippocampal neurons of convulsive rats. METHODS: The acute convulsion model was induced by intraperitoneal injection of pentylenetetrazol in SD rats who were then randomized into model group and acupuncture group. Rats of the acupuncture group received manual acupuncture stimulation of "Baihui" (GV20) and "Dazhui" (GV14) for 30 min, once daily for 7 days. Then, the blood samples taken from the abdominal aorta of rats in the convulsion model and acupuncture groups were processed into serum samples, i.e. non-acupuncture serum and acupuncture se-rum. The primary-cultured hippocampal neurons of fetal rats were cultured for 10 days and then divided into normal extracellular fluid (normal) group, magnesium (Mg2+) free extracellular fluid group, acupuncture serum group and non-acupuncture serum group. At the 10th day, the neurons in the normal group were cultured continuously in extracellular fluid for 3 h, and then cultured in DMEM/F12(1∶1) medium (planting fluid); neurons in the Mg2+ free group were cultured in magnesium-free fluid medium to induce epileptic-like discharge; neurons in the acupuncture serum group were cultured in the mixed medium of planting fluid and 10% acupuncture serum; and neurons in the non-acupuncture serum were cultured in the mixed culture medium of planting fluid and non-acupuncture serum (10%). At last, these neurons in the above-mentioned groups were cultured in the magnesium-free extracellular fluid continuously for 2, 12 and 48 h, respectively, followed by detecting the expression levels of MAP-2 and GAP-43 proteins at the 3 time points by using immunofluorescence and Western blot, separately. RESULTS: The rate of MAP-2 positive cells and protein expression at 2, 12 and 48 h, and the rate of GAP-43 positive cells and protein expression at 12 and 48 h in the hippocampal neurons were significantly down-regulated in the Mg2+ free group in contrast to the normal group (P<0.05，P<0.01). Compared to the Mg2+ free group, the rates of MAP-2 and GAP-43 positive cells and protein expression at 2, 12 and 48 h were considerably up-regulated in the acupuncture serum group (P<0.05，P<0.01), but not in the non-acupuncture serum group (P>0.05). CONCLUSION: Acupuncture serum can significantly up-regulate the expression of MAP-2 and GAP-43 proteins in hip-pocampal neurons, which may play a positive role in improving synaptic plasticity and neuronal damage in convulsion rats.

Apigenin Induces Autophagy and Cell Death by Targeting EZH2 under Hypoxia Conditions in Gastric Cancer Cells.

Hypoxia is a major obstacle to gastric cancer (GC) therapy and leads to chemoresistance as GC cells are frequently exposed to the hypoxia environment. Apigenin, a flavonoid found in traditional medicine, fruits, and vegetables and an HDAC inhibitor, is a powerful anti-cancer agent against various cancer cell lines. However, detailed mechanisms involved in the treatment of GC using APG are not fully understood. In this study, we investigated the biological activity of and molecular mechanisms involved in APG-mediated treatment of GC under hypoxia. APG promoted autophagic cell death by increasing ATG5, LC3-II, and phosphorylation of AMPK and ULK1 and down-regulating p-mTOR and p62 in GC. Furthermore, our results show that APG induces autophagic cell death via the activation of the PERK signaling, indicating an endoplasmic reticulum (ER) stress response. The inhibition of ER stress suppressed APG-induced autophagy and conferred prolonged cell survival, indicating autophagic cell death. We further show that APG induces ER stress- and autophagy-related cell death through the inhibition of HIF-1α and Ezh2 under normoxia and hypoxia. Taken together, our findings indicate that APG activates autophagic cell death by inhibiting HIF-1α and Ezh2 under hypoxia conditions in GC cells.

Cycloastragenol alleviates airway inflammation in asthmatic mice by inhibiting autophagy.

Cycloastragenol (CAG), a secondary metabolite from the roots of Astragalus zahlbruckneri, has been reported to exert anti­inflammatory effects in heart, skin and liver diseases. However, its role in asthma remains unclear. The present study aimed to investigate the effect of CAG on airway inflammation in an ovalbumin (OVA)­induced mouse asthma model. The current study evaluated the lung function and levels of inflammation and autophagy via measurement of airway hyperresponsiveness (AHR), lung histology examination, inflammatory cytokine measurement and western blotting, amongst other techniques. The results demonstrated that CAG attenuated OVA­induced AHR in vivo. In addition, the total number of leukocytes and eosinophils, as well as the secretion of inflammatory cytokines, including interleukin (IL)­5, IL­13 and immunoglobulin E were diminished in bronchoalveolar lavage fluid of the OVA­induced murine asthma model. Histological analysis revealed that CAG suppressed inflammatory cell infiltration and goblet cell secretion. Notably, based on molecular docking simulation, CAG was demonstrated to bind to the active site of autophagy­related gene 4­microtubule­associated proteins light chain 3 complex, which explains the reduced autophagic flux in asthma caused by CAG. The expression levels of proteins associated with autophagy pathways were inhibited following treatment with CAG. Taken together, the results of the present study suggest that CAG exerts an anti­inflammatory effect in asthma, and its role may be associated with the inhibition of autophagy in lung cells.

Treatment of Parkinson's disease in Zebrafish model with a berberine derivative capable of crossing blood brain barrier, targeting mitochondria, and convenient for bioimaging experiments.

Berberine is a famous alkaloid extracted from Berberis plants and has been widely used as medications and functional food additives. Recent studies reveal that berberine exhibits neuroprotective activity in animal models of Parkinson's disease (PD), the second most prevalent neurodegenerative disorders all over the world. However, the actual site of anti-PD action of berberine remains largely unknown. To this end, we employed a fluorescently labeled berberine derivative BBRP to investigate the subcellular localization and blood brain barrier (BBB) permeability in a cellular model of PD and zebrafish PD model. Biological investigations revealed that BBRP retained the neuroprotective activity of berberine against PD-like symptoms in PC12 cells and zebrafish, such as protecting 6-OHDA induced cell death, relieving MPTP induced PD-like behavior and increasing dopaminergic neuron loss in zebrafish. We also found that BBRP could readily penetrate BBB and function in the brain of zebrafish suffering from PD. Subcellular localization study indicated that BBRP could rapidly and specifically accumulate in mitochondria of PC12 cells when it exerted anti-PD effect. In addition, BBRP could suppress accumulation of Pink1 protein and inhibit the overexpression of LC3 protein in 6-OHDA damaged cells. All these results suggested that the potential site of action of berberine is mitochondria in the brain under the PD condition. Therefore, the findings described herein would be useful for further development of berberine as an anti-PD drug.

Hippocampal and Reticulo-Thalamic Parvalbumin Interneurons and Synaptic Re-Organization during Sleep Disorders in the Rat Models of Parkinson's Disease Neuropathology.

We investigated the alterations of hippocampal and reticulo-thalamic (RT) GABAergic parvalbumin (PV) interneurons and their synaptic re-organizations underlying the prodromal local sleep disorders in the distinct rat models of Parkinson's disease (PD). We demonstrated for the first time that REM sleep is a predisposing state for the high-voltage sleep spindles (HVS) induction in all experimental models of PD, particularly during hippocampal REM sleep in the hemiparkinsonian models. There were the opposite underlying alterations of the hippocampal and RT GABAergic PV+ interneurons along with the distinct MAP2 and PSD-95 expressions. Whereas the PD cholinopathy enhanced the number of PV+ interneurons and suppressed the MAP2/PSD-95 expression, the hemiparkinsonism with PD cholinopathy reduced the number of PV+ interneurons and enhanced the MAP2/PSD-95 expression in the hippocampus. Whereas the PD cholinopathy did not alter PV+ interneurons but partially enhanced MAP2 and suppressed PSD-95 expression remotely in the RT, the hemiparkinsonism with PD cholinopathy reduced the PV+ interneurons, enhanced MAP2, and did not change PSD-95 expression remotely in the RT. Our study demonstrates for the first time an important regulatory role of the hippocampal and RT GABAergic PV+ interneurons and the synaptic protein dynamic alterations in the distinct rat models of PD neuropathology.

Sex-Specific Differences in Autophagic Responses to Experimental Ischemic Stroke.

Ischemic stroke triggers a series of complex pathophysiological processes including autophagy. Differential activation of autophagy occurs in neurons derived from males versus females after stressors such as nutrient deprivation. Whether autophagy displays sexual dimorphism after ischemic stroke is unknown. We used a cerebral ischemia mouse model (middle cerebral artery occlusion, MCAO) to evaluate the effects of inhibiting autophagy in ischemic brain pathology. We observed that inhibiting autophagy reduced infarct volume in males and ovariectomized females. However, autophagy inhibition enhanced infarct size in females and in ovariectomized females supplemented with estrogen compared to control mice. We also observed that males had increased levels of Beclin1 and LC3 and decreased levels of pULK1 and p62 at 24 h, while females had decreased levels of Beclin1 and increased levels of ATG7. Furthermore, the levels of autophagy markers were increased under basal conditions and after oxygen and glucose deprivation in male neurons compared with female neurons in vitro. E2 supplementation significantly inhibited autophagy only in male neurons, and was beneficial for cell survival only in female neurons. This study shows that autophagy in the ischemic brain differs between the sexes, and that autophagy regulators have different effects in a sex-dependent manner in neurons.