Inhibition of hepatitis B virus through PPAR-JAK/STAT pathway modulation by electroacupuncture and tenofovir disoproxil fumarate combination therapy.

BACKGROUND: Acupuncture combined with nucleos(t)ide analogues (NAs) has shown promise in treating chronic hepatitis B (CHB), though mechanisms remain unclear. This study evaluates the antiviral effects of combining acupuncture with NAs against hepatitis B virus (HBV) and explores underlying mechanisms. METHODS: The HBV-infected mouse model, established using the high-pressure hydrodynamic method, was divided into three groups: normal saline (NS), tenofovir disoproxil fumarate (TF), and electroacupuncture combined with TF (E_T), n = 6. Antiviral effects were assessed by monitoring HBV DNA, HBsAg, and HBeAg levels weekly. Mechanistic insights were gained via transcriptomics, metabolomics, and 16S rDNA sequencing, validated by WB, PCR, and flow cytometry. RESULTS: Serum HBV DNA levels decreased by 1.98 log10 IU/mL in TF and 2.2 log10 IU/mL in E_T groups compared to NS. Serum HBeAg decreased by 10.61 % in TF and 35.75 % in E_T, while HBsAg decreased by 7.38 % and 37.58 %, respectively. Multi-omics indicated E_T modulates the PPAR pathway, upregulates taurine and all-trans-retinoic acid, and increases gut microbiota like Bacteroides and Blautia. E_T also enhanced tight junction proteins (ZO-1, Occludin, Claudin-4), improving intestinal barrier integrity. Mechanistically, E_T inhibited the PGC-1α/PPAR-α/SIRT1 pathway, reducing PGC-1α, PPAR-α, SIRT1, RXRα, and HNF4α, while promoting JAK/STAT signaling via IFN-γ, p-JAK1, p-JAK2, p-STAT1, IRF8, and suppressing SOCS-1. CONCLUSION: E_T more effectively inhibited HBV replication, showing superior antigen inhibition, particularly HBsAg, than TF alone. This may be due to PPAR-JAK/STAT pathway regulation, suggesting E_T as a potential adjuvant therapy for CHB, especially in achieving a functional cure.

Non-coding RNA and its network in the pathogenesis of Myasthenia Gravis.

Myasthenia Gravis (MG) is a chronic autoimmune disease that primarily affects the neuromuscular junction, leading to muscle weakness in patients with this condition. Previous studies have identified several dysfunctions in thymus and peripheral blood mononuclear cells (PBMCs), such as the formation of ectopic germinal centers in the thymus and an imbalance of peripheral T helper cells and regulatory T cells, that contribute to the initiation and development of MG. Recent evidences suggest that noncoding RNA, including miRNA, lncRNA and circRNA may play a significant role in MG progression. Additionally, the network between these noncoding RNAs, such as the competing endogenous RNA regulatory network, has been found to be involved in MG progression. In this review, we summarized the roles of miRNA, lncRNA, and circRNA, highlighted their potential application as biomarkers in diagnosing MG, and discussed their potential regulatory networks in the abnormal thymus and PBMCs during MG development.

An Experimental Animal Study: Electroacupuncture Facilitates Antiviral Immunity Against Hepatitis B Virus Through the IFN-γ/JAK/STAT Axis.

Background: Chronic hepatitis B (CHB) remains a global health challenge, necessitating innovative therapeutic strategies. Enhancing the body's immune response against the hepatitis B virus (HBV) emerges as a fundamental strategy for achieving a functional cure. While acupuncture has shown potential in immune modulation, its specific anti-HBV effects are not well understood. This study evaluates the potential of electroacupuncture (EA) in HBV infection and explores its underlying immunological mechanisms using a mouse model. Methods: HBV-infected mice were established using the high-pressure hydrodynamic method and divided into four groups: normal saline (NS), EA, sham EA (SE), and tenofovir disoproxil fumarate (TF), with n = 6 per group. During treatment, blood was collected every Sunday via the orbital sinus to monitor HBV DNA, HBsAg, and HBeAg levels. Transcriptomics and metabolomics analyses were employed to unearth clues regarding EA's anti-HBV mechanism. Validation of these mechanisms included splenic T-cell flow analysis, Western blotting, RT-qPCR, immunofluorescence, and ELISA. Results: Serum HBV DNA levels decreased by 1.10, 0.19, and 1.98 log10 IU/mL in the EA, SE, and TF-treated mice, respectively, compared to the NS. Concurrently, the hepatic HBV DNA levels decreased by 1.09, 0.24, and 2.03 log10 IU/mL. EA also demonstrated superior inhibition of HBV antigens, with serum HBeAg levels decreasing by 43.86%, 8.74%, and 8.03%, and serum HBsAg levels decreasing by 28.01%, 0.26%, and 9.39% in the EA, SE, and TF groups, respectively. Further analysis through transcriptomics and metabolomics revealed that EA's anti-HBV effects primarily hinge on immune modulation, particularly the IFN-γ/JAK/STAT pathway and taurine metabolism. EA also increased the ratio of splenic CD8+ CD69+ and CD8+ IFN-γ+ T-cells while upregulating key proteins in the JAK/STAT pathway and cytokines associated with antiviral immunity. Conclusion: EA manifests inhibitory effects on HBV, particularly in antigen suppression, with its mode of action intricately linked to the regulation of IFN-γ/JAK/STAT.

Pilot implementation of the revised criteria for staging of Alzheimer's disease by the Alzheimer's Association Workgroup in a tertiary memory clinic.

INTRODUCTION: We aimed to evaluate the feasibility of the 2024 Alzheimer's Association Workgroup's integrated clinical-biological staging scheme in outpatient settings within a tertiary memory clinic. METHODS: The 2018 syndromal cognitive staging system, coupled with a binary biomarker classification, was implemented for 236 outpatients with cognitive concerns. The 2024 numeric clinical staging framework, incorporating biomarker staging, was specifically applied to 154 individuals within the Alzheimer's disease (AD) continuum. RESULTS: The 2024 staging scheme accurately classified 95.5% AD. Among these, 56.5% exhibited concordant clinical and biological stages (canonical), 34.7% demonstrated more advanced clinical stages than biologically expected (susceptible), and 8.8% displayed the inverse pattern (resilient). The susceptible group was characterized by a higher burden of neurodegeneration and inflammation than anticipated from tau, whereas the resilient group showed the opposite. DISCUSSION: The 2024 staging scheme is generally feasible. A discrepancy between clinical and biological stages is relatively frequent among symptomatic patients with AD. HIGHLIGHTS: The 2024 AA staging scheme is generally feasible in a tertiary memory clinic. A discrepancy between clinical and biological stages is relatively frequent in AD. The mismatch may be influenced by a non-specific pathological process involved in AD. Individual profiles like aging and lifestyles may contribute to such a mismatch. Matched and mismatched cases converge toward similar clinical outcomes.

Electroacupuncture improves allodynia and central sensitization via modulation of microglial activation associated P2X4R and inflammation in a rat model of migraine.

Background: Recent studies have demonstrated that activated microglia were involved in the pathogenesis of central sensitization characterized by cutaneous allodynia in migraine. Activation of microglia is accompanied by increased expression of its receptors and release of inflammatory mediators. Acupuncture and its developed electroacupuncture (EA) have been recommended as an alternative therapy for migraine and are widely used for relieving migraine-associated pain. However, it remains rare studies that show whether EA exerts anti-migraine effects via inhibiting microglial activation related to a release of microglial receptors and the inflammatory pathway. Therefore, this study aimed to investigate EA' ability to ameliorate central sensitization via modulation of microglial activation, microglial receptor, and inflammatory response using a rat model of migraine induced by repeated epidural chemical stimulation. Methods: In the present study, a rat model of migraine was established by epidural repeated inflammatory soup (IS) stimulation and treated with EA at Fengchi (GB20) and Yanglingquan (GB34) and acupuncture at sham-acupoints. Pain hypersensitivity was further determined by measuring the mechanical withdrawal threshold using the von-Frey filament. The changes in c-Fos and ionized calcium binding adaptor molecule 1 (Ibal-1) labeled microglia in the trigeminal nucleus caudalis (TNC) were examined by immunflurescence to assess the central sensitization and whether accompanied with microglia activation. In addition, the expression of Ibal-1, microglial purinoceptor P2X4, and its associated inflammatory signaling pathway mediators, including interleukin (IL)-1ß, NOD-like receptor protein 3 (NLRP3), and Caspase-1 in the TNC were investigated by western blot and real-time polymerase chain reaction analysis. Results: Allodynia increased of c-Fos, and activated microglia were observed after repeated IS stimulation. EA alleviated the decrease in mechanical withdrawal thresholds, reduced the activation of c-Fos and microglia labeled with Ibal-1, downregulated the level of microglial purinoceptor P2X4, and limited the inflammatory response (NLRP3/Caspase-1/IL-1ß signaling pathway) in the TNC of migraine rat model. Conclusions: Our results indicate that the anti-hyperalgesia effects of EA ameliorate central sensitization in IS-induced migraine by regulating microglial activation related to P2X4R and NLRP3/IL-1ß inflammatory pathway.

Increased Cerebral Level of P2X7R in a Tauopathy Mouse Model by PET Using [18F]GSK1482160.

Neuroinflammation plays an important role in Alzheimer's disease and primary tauopathies. The aim of the current study was to map [18F]GSK1482160 for imaging of purinergic P2X7R in Alzheimer's disease and primary tauopathy mouse models. Small animal PET was performed using [18F]GSK1482160 in widely used mouse models of Alzheimer's disease (APP/PS1, 5×FAD, and 3×Tg), 4-repeat tauopathy (rTg4510) mice, and age-matched wild-type mice. Increased uptake of [18F]GSK1482160 was observed in the brains of 7-month-old rTg4510 mice compared to wild-type mice and compared to 3-month-old rTg4510 mice. A positive correlation between hippocampal tau [18F]APN-1607 and [18F]GSK1482160 uptake was found in rTg4510 mice. No significant differences in the uptake of [18F]GSK1482160 was observed for APP/PS1 mice, 5×FAD mice, or 3×Tg mice. Immunofluorescence staining further indicated the distribution of P2X7Rs in the brains of 7-month-old rTg4510 mice with accumulation of tau inclusion. These findings provide in vivo imaging evidence for an increased level of P2X7R in the brains of tauopathy mice.

Multicellular ecotypes shape progression of lung adenocarcinoma from ground-glass opacity toward advanced stages.

Lung adenocarcinoma is a type of cancer that exhibits a wide range of clinical radiological manifestations, from ground-glass opacity (GGO) to pure solid nodules, which vary greatly in terms of their biological characteristics. Our current understanding of this heterogeneity is limited. To address this gap, we analyze 58 lung adenocarcinoma patients via machine learning, single-cell RNA sequencing (scRNA-seq), and whole-exome sequencing, and we identify six lung multicellular ecotypes (LMEs) correlating with distinct radiological patterns and cancer cell states. Notably, GGO-associated neoantigens in early-stage cancers are recognized by CD8+ T cells, indicating an immune-active environment, while solid nodules feature an immune-suppressive LME with exhausted CD8+ T cells, driven by specific stromal cells such as CTHCR1+ fibroblasts. This study also highlights EGFR(L858R) neoantigens in GGO samples, suggesting potential CD8+ T cell activation. Our findings offer valuable insights into lung adenocarcinoma heterogeneity, suggesting avenues for targeted therapies in early-stage disease.

Electroacupuncture Reduces Inflammatory Bowel Disease in Obese Mice by Activating the Nrf2/HO-1 Signaling Pathways and Repairing the Intestinal Barrier.

Background: Electroacupuncture (EA) is used to treat inflammatory bowel disease (IBD). Nevertheless, the precise mechanisms by which this approach safeguards against obesity-induced intestinal barrier damage has not been fully understood. Objective: This study aimed to assess whether EA could ameliorate intestinal barrier damage that had been reversed in a mouse model of obesity induced by a high-fat diet (HFD) and whether this repair is correlated with ferroptosis and gut microbiota enhancement. Methods: To assess the potential of EA to prevent obesity and restore the intestinal barrier, we divided in C57BL/6J mice into two groups; one was fed with HFD and another one with a normal diet. Samples of stool, blood, fat, and intestinal epithelium were then evaluated, along with body weight. Results: Following EA, we observed a significant reduction in body weight, fat accumulation, and serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels; an increase was seen in high-density lipoprotein cholesterol (HDL-C) levels. EA also activated the Nrf2 signaling pathway; upregulated the expression of GPX4, FTH1, and SLC7A11; and downregulated the expression of TFR1. In addition, the administration of EA resulted in a notable modification of the gut microbiota composition, characterized by a decrease in the Firmicutes to Bacteroidetes ratio. Conclusion: EA had beneficial effects on weight loss and showed potential ability to repair the intestinal barrier by activating the Nrf2 signaling pathway, inhibiting intestinal inflammation and ferroptosis, and regulating the intestinal microbiota to treat IBD caused by HFD-induced obesity.

Electroacupuncture at Fengchi(GB20) and Yanglingquan(GB34) Ameliorates Paralgesia through Microglia-Mediated Neuroinflammation in a Rat Model of Migraine.

BACKGROUND: Multiple studies have suggested that paralgesia (hyperalgesia and cutaneous allodynia) in migraine reflects the activation and sensitisation of the trigeminovascular system (TGVS). In particular, it reflects the second-order and higher nerve centre sensitisation, which is caused and maintained by neuroinflammation. Microglia activation leads to the release of proinflammatory cytokines involved in inflammatory responses. Accumulating evidence indicates that electroacupuncture (EA) is effective in ameliorating paralgesia, but the underlying mechanisms of EA in migraine attacks caused by microglia and microglia-mediated inflammatory responses are still unclear. The purpose of this study was to explore whether EA could ameliorate the dysregulation of pain sensation by suppressing microglial activation and the resulting neuroinflammatory response, and to evaluate whether this response was regulated by Toll-like receptor 4 (TLR4)/nuclear factor-kappa B(NF-κB) in the trigeminal nucleus caudalis (TNC) in a rat model of migraine. METHODS: Repeated Inflammatory Soup (IS) was infused into the dura for seven sessions to establish a recurrent migraine-like rat model, and EA treatment was administered at Fengchi (GB20) and Yanglingquan (GB34) after daily IS infusion. Facial mechanical withdrawal thresholds were measured to evaluate the change in pain perception, and plasma samples and the TNC tissues of rats were collected to examine the changes in calcitonin gene-related peptide (CGRP), the Ibal-1-labelled microglial activation, and the resulting inflammatory response, including interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), and their regulatory molecules TLR4/NF-κB, via enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and Western blot analysis. RESULTS: Repeated IS injections into the dura induced facial mechanical paralgesia, which is the manifestation of migraine attacks, and increased the expression of CGRP, Ibal-1, microglial mediated inflammatory cytokines (IL-1ß, TNF-α, IL-6), and regulatory molecules TLR4/NF-κB. EA at GB20/34 significantly attenuated repetitive IS-induced pain hypersensitivity. This effect was consistent with decreased levels of CGRP and inflammatory cytokines in the plasma and the TNC via the inhibition of microglia activation, and this response may be regulated by TLR4/NF-κB. CONCLUSIONS: EA ameliorated paralgesia in repetitive IS-induced migraine-like rats, which was mainly mediated by a reduction in microglial activation and microglial-mediated inflammatory responses that could be regulated by TLR4/NF-κB.

Viscosity & SO2-sensitive dual colorimetric effect fluorescent sensor enabled imaging detection within plant onion and biological system.

The biological microenvironment includes important parameters such as viscosity, polarity, temperature, oxygen content and pH. In particular, abnormal cell viscosity is associated with the development of major diseases. Sulphur dioxide (SO2) serves not only as an essential atmospheric pollutant but also an influential signalling molecule in biological cells, predisposing individuals to increased respiratory disease. In this work, we designed and synthesized a novel fluorescent probe CouCN-V&S with dual response to micro environmental viscosity and SO2. The probe monitored viscosity and SO2 separately through dual emission channels with a difference of 135 nm. The probe responded sensitively to SO2 (<1s) and exhibited satisfactory immunity to interference and pH stability. The probe was successfully applied to imaging cellular, intra-zebrafish viscosity and SO2 changes. Interestingly, we took onion epidermal cells as model and explored the capability of probe CouCN-V&S to image SO2 in plant cells for the first time.

The Fractal Network Structure of Silk Fibroin Molecules and Its Effect on Spinning of Silkworm Silk.

Animal silk is usually considered to exist as a solid fiber with a highly ordered structure, formed by the hierarchical assembly starting from a single silk fibroin (SF) chain. However, this study showed that silk protein molecules existed in the form of a fractal network structure in aqueous solution, rather than as a single chain. This type of network was relatively rigid with low fractal dimension. Finite element analysis revealed that this network structure significantly helped in the stable storage of SF prior to the spinning process and in the rapid formation of a ß-sheeted nanocrystalline and nematic texture during spinning. Further, the strong but brittle mechanical properties of Bombyx mori silk could also be well-explained through the fractal network model of silk fibroin. The strength was mainly derived from the dual network structure, consisting of nodes and ß-sheet cross-links, whereas the brittleness could be attributed to the rigidity of the SF chains between these nodes and cross-links. In summary, this study presents insights from network topology for understanding the spinning process of natural silk and the structure-property relationship in silk materials.

Effect of different substituents on the fluorescence properties of precursors of synthetic GFP analogues and a polarity-sensitive lipid droplet probe with AIE properties for imaging cells and zebrafish.

The green fluorescent protein (GFP) is a purely natural specialty protein that has been widely used to design synthetic fluorescent probes. In the present work we designed and synthesized a series of fluorescent compounds akin to GFP precursors by a one-step method, and investigated the luminescence properties of the fluorescent compounds by varying the substituents. We presented the first systematic summary of the photophysical data including extinction coefficients and fluorescence quantum yields for this class of fluorescent dyes. We also carried out density functional theory (DFT) calculations for these dyes to investigate the effect of electronic effects due to different substituents. These studied optical properties may provide a reference for later probe design. More interestingly, we have developed a polarity-sensitive lipid droplet probe T-LD with AIE properties on this basis. The probe exhibited not only favorable pH stability and kinetic stability in terms of optical properties, but also solvent discolouration and polarity-sensitive properties, and was able to label intracellular lipid droplets. We successfully applied the probe for intracellular lipid droplet level monitoring and zebrafish imaging.

Electroacupuncture Alleviates Depressive-like Behavior by Modulating the Expression of P2X7/NLRP3/IL-1ß of Prefrontal Cortex and Liver in Rats Exposed to Chronic Unpredictable Mild Stress.

Depression is a complex clinical disorder associated with poor outcomes. Electroacupuncture (EA) has been demonstrated to have an important role in both clinical and pre-clinical depression investigations. Evidence has suggested that the P2X7 receptor (P2X7R), NLRP3, and IL-1ß play an important role in depressive disorder. Our study is aimed at exploring the role of EA in alleviating depression-like behaviors in rats. We therefore investigated the effects of EA on the prefrontal cortex and liver of rats subjected to chronic unpredictable mild stress (CUMS) through behavior tests, transmission electron microscopy, Nissl staining, HE staining, immunohistochemistry and Western blotting. Five weeks after exposure to CUMS, Sprague-Dawley (SD) rats showed depression-like behavior. Three weeks after treatment with brilliant blue G (BBG) or EA, depressive symptoms were significantly improved. Liver cells and microglia showed regular morphology and orderly arrangement in the BBG and EA groups compared with the CUMS group. Here we show that EA downregulated P2X7R/NLRP3/IL-1ß expression and relieved depression-like behavior. In summary, our findings demonstrated the efficacy of EA in alleviating depression-like behaviors induced by CUMS in rats. This suggests that EA may serve as an adjunctive therapy in clinical practice, and that P2X7R may be a promising target for EA intervention on the liver-brain axis in treatment of depression.

Viscosity-Sensitive Solvatochromic Fluorescent Probes for Lipid Droplets Staining.

Lipid droplets (LDs) are simple intracellular storage sites for neutral lipids and exhibit important impact on many physiological processes. For example, the changes in the polar microenvironment inside LDs could affect physiological processes, such as lipid metabolism and storage, protein degradation, signal transduction, and enzyme catalysis. Herein, a new fluorescent chemo-sensor (Couoxo-LD) was formulated by our molecular design strategy. The probe could be applied to effectively label intracellular lipid droplets. Intriguingly, Couoxo-LD demonstrated positive sensitivity to both polarity and viscosity, which might be attributed to its D-π-A structure and the twisted rotational behavior of the carbon-carbon double bond (TICT). Additionally, Couoxo-LD was successfully implemented in cellular imaging due to its excellent selectivity, pH stability, and low biotoxicity. In HeLa cells, the co-localization curve between Couoxo-LD and commercial lipid droplet dyes overlapped at 0.93. The results indicated that the probe could selectively sense LDs in HeLa cells. Meanwhile, Couoxo-LD can be applied for in vivo imaging of zebrafish.

[Effect of electroacupuncture on cognitive impairment in APP/PS1 mice based on TLR4/NF-κB/NLRP3 pathway].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expressions of tight junction related proteins Claudin-5, ZO-1 in the colon and hippocampus, Toll-like receptor 4/nuclear factor-kappa B/NOD-like receptor protein 3 (TLR4/NF-κB/NLRP3) pathway in the hippocampus of APP/PS1 mice, so as to explore its mechanisms underlying improvement of cognitive impairment. METHODS: Eighteen 5-month-old male APP/PS1 mice were equally randomized into model and EA groups,and nine 5-month-old male C57BL/6 mice were used as the normal control. EA(2 Hz, 1 mA) was applied to "Baihui" (GV20), "Dachangshu" (BL25) and "Zusanli" (ST36) for 15 min, once daily, 5 days a week for 5 weeks. The Morris water maze swimming test was used to evaluate the mice's cognitive impairment. Nissl staining was used to observe the pathological morphology of hippocampus. The expression of amyloid ß-peptide (Aß) in brain tissue was detect by immunohistochemistry; the contents of lipopolysaccharide (LPS) in colon, serum and hippocampus were detected by ELISA; the expression levels of Claudin-5, ZO-1 in colon and hippocampus, and TLR4/NF-κB/NLRP3 pathway related proteins in hippocampus were detected by Western blot. RESULTS: Compared with the normal group, the escape latency of the mice in the model group was prolonged from the 3rd day (P<0.05, P<0.01), the number of crossing the platform and the percentage of target quadrant residence time were significantly decreased (P<0.01), and the contents of LPS in colon, serum and hippocampus were significantly increased (P<0.01), the expression levels of TLR4, NF-κB p65, NLRP3, Caspase-1, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in hippocampus and Aß in brain tissue were significantly increased (P<0.01), while the expression levels of Claudin-5, ZO-1 in colon and hippocampus were significantly decreased (P<0.01). Compared with the model group, the escape latency of mice in the EA group was shortened from the 4th day (P<0.05, P<0.01), the number of crossing the platform and the percentage of target quadrant residence time were increased (P<0.01, P<0.05), and the contents of LPS in serum and hippocampus were decreased (P<0.05), and the expression levels of TLR4, NF-κB p65, Caspase-1, NLRP3, IL-1ß, TNF-α in hippocampus and Aß in brain tissue were significantly decreased (P<0.05, P<0.01), while the expression levels of Claudin-5, ZO-1 in colon and hippocampus were significantly increased (P<0.05, P<0.01). Outcomes of Nissl staining showed dispersed arrangement of neurons with nuclear pyknosis or hyperchromasia in the hippocampus, and a decreased number of cell layers in the model group, which was relatively milder in the EA group. CONCLUSION: EA may improve the cognitive impairment of APP/PS1 mice by up-regulating the expression of Claudin-5 and ZO-1, reducing the transposition of gut-derived LPS to the central nervous system, inhibiting the over-activation of TLR4/NF-κB/NLRP3 pathway, and alleviating the inflammatory reaction of the central nervous system.

[Effect of electroacupuncture on hepatocyte autophagy and oxidative stress in SAMP8 mice by regulating AMPK/mTOR/ULK1 signaling pathway].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on physical strength and expression levels of hepatic AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), unc-51 like autophagy activating kinase 1 (ULK1) proteins and Atg5, Atg7, Atg13, Beclin1 and ULK1 mRNAs in aging (senescence accelerated mouse/prone 8, SAMP8)mice, so as to exp lore its mechanism underlying delaying aging by activating AMPK/mTOR/ULK1 signaling pathway. METHODS: Twenty-four male SAMP8 mice were randomly divided into model group, rapamycin (autophagy inducer) group, EA group and EA+autophagy inhibitor (EA+inhibitor) group, with 6 mice in each group, and 6 homologous anti-rapid aging male (SAMR1) mice in the same age were used as the control group. Mice of the rapamycin group received intraperitoneal injection of rapamycin solution (2 mg·kg-1·d-1). EA (2 Hz, 1 mA) was applied to bilateral "Taichong"(LR3)and "Shenshu"(BL23) for 15 min each time. Mice of the EA+inhibitor group received intraperitoneal injection of mTOR inhibitor 3-methyladenine (1.5 mg·kg-1·d-1) before the EA intervention each time. The above-mentioned interventions were conducted 6 times a week for 2 consecutive weeks. Physical conditions of mice were assessed by exhaustive swimming tests. Histopathological changes of the liver were observed by H.E. staining. Western blot was used to detect the expression of AMPK, phosphorylated AMPK (p-AMPK), mTOR, phosphorylated mTOR (p-mTOR), ULK1 and phosphorylated ULK1 (p-ULk1) in the liver tissues. The expression levels of Atg5, Atg7, Atg13, Beclin1 and ULK1 (cellular autophagy-related genes) mRNAs in the liver were detected by quantitative real-time PCR. The immunoactivity (IA) of heme oxygenase 1 (HO-1) in the liver was detected by immunohistochemistry, and the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) of the liver were measured by hydroxylamine method for assessing the level of oxidative stress. RESULTS: Compared with the control group, the duration of exhaustive swimming, the expression levels of AMPK, p-AMPK, ULK1 and p-ULK1 proteins, and Atg5, Atg7, Atg13, Beclin1 and ULK1 mRNA, HO-1 IA and SOD activity were considerably down-regulated (P<0.01), while the expression levels of mTOR and p-mTOR and MDA content were significantly up-regulated (P<0.01) in the model group. In comparison with the model group, the duration of the exhausted swimming, the expression levels of AMPK, p-AMPK, ULK1 and p-ULK1 proteins, and Atg5, Atg7, Atg13, Beclin1 and ULK1 mRNAs, HO-1 IA and SOD activity were significantly up-regulated (P<0.01, P<0.05), whereas the expression levels of mTOR and p-mTOR proteins and MDA content were notably down-regulated (P<0.01, P<0.05) in the rapamycin, EA and EA+inhibitor groups. The improvement of the abovementioned indexes of EA+inhibitor group was not as good as rapamycin and EA groups (P<0.01), suggesting an elimination of the therapeutic effects after administration of 3-methyladenine. No significant differences were found between the rapamycin and EA groups in the abovementioned indexes (P>0.05) except p-mTOR and mTOR which were higher in the EA group (P<0.01). H.E. staining showed ambiguous boundary of the liver lobule, disordered arrangement of hepatocytes with a large amount of fat vacuoles at different size and deviation of nucleus, and lysis of some hepatocytes. These situations were relatively milder in the rapamycin and EA groups. CONCLUSION: EA may enhance physical strength and promote cellular autophagy in the liver of aging mice by regulating AMPK/mTOR/ULK1 signaling, thereby inhibiting excessive oxidative stress, and delaying aging process to some extent.

[Effects of electroacupuncture at "Jiaji"(EX-B2) on autophagy and endoplasmic reticulum stress in spinal cord injury mice].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Jiaji" (EX-B2) on the levels of autophagy and endoplasmic reticulum stress in mice with spinal cord injury (SCI), so as to explore its mechanism underlying improvement of SCI. METHODS: A total of 60 female C57BL/6 mice were randomly divided into sham operation, model and EA groups， which were further divided into 7 d and 14 d subgroups (10 mice in each subgroup). The SCI model was established by pressing the exposed spinal cord (L1) with a vascular clamp for 15 s. EA was applied to bilateral EX-B2 3 h after modeling, once a day for 7 and 14 d, respectively. Basso Mouse Scale(BMS) for locomotion was used to evaluate hindlimb motor function on day 7 and 14 after SCI. H.E. staining was used to observe histopathologic changes of the injured spinal cord tissue, and Western blot employed to detect the expression of glucose regulatory protein-78 (GRP78), Caspase-12, microtubule-associated protein light chain 3 II (LC-II) and P62(also known as sqstm1/Sequestome1) proteins. Immunofluorescence staining was used to detect the immunoacti-vities of spinal CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP, an endoplasmic reticulum stress-inducible protein) and P62. RESULTS: On the 7th and 14th day after SCI, the BMS scores and expression levels of LC3II protein were significantly down-regulated (P<0.05), and the expression levels of P62, GRP78 and Caspase-12 proteins, the immunoactivities of CHOP and P62 were all significantly up-regulated on both day 7 and 14 in the model group than in the sham operation group (P<0.05).Compared with the model group, the BMS scores and the expression levels of LC3II protein were significantly increased on both day 7 and 14 (P<0.05), while the expression levels of P62, GRP78 and Caspase-12 proteins, and the immunoactivities of CHOP and P62 were obviously decreased on day 7 and 14 in the EA group (P<0.05). Outcomes of H.E. stain showed that the cells with nuclei pyknosis and swelling and the necrotic cells appeared in the model group, which was relatively fewer in the EA group. CONCLUSION: EA of EX-B2 can improve the locomotor function in SCI mice, which may be related to its effects in up-regulating the expression of LC3II (to promote cell autophagy), and down-regulating the expression of P62, GRP78, Caspase-12 and CHOP proteins (to inhibit endoplasmic reticulum stress) in the spinal cord tissue.

[Effect of electroacupuncture combined with Donepezil on learning-memory ability and expression of hippocampal ß-amyloid clearance-related genes in SAMP8 mice].

OBJECTIVE: To investigate the effect of electroacupuncture (EA) combined with Donepezil on learning-memory ability and gene expression of ß-amyloid (Aß) clearance-related factors in the hippocampus in senescence-accelerated mouse prone 8 (SAMP8) mice, so as to explore their synthetic effect in improving dementia of Alzheimer's disease (AD)．. METHODS: Male SAMP8 mice (30-week-old) were randomly divided into model, medication and EA＋medication groups (n＝6 mice in each group), and other 6 senescence-resistant 1 (SAMR1) mice were used as the control group. Mice of the medication and EA＋medication group received gavage of Donepezil (1.3 mg•kg－1•d－1) once daily for 4 weeks. EA (2 Hz, 1 mA) was applied to "Baihui"(GV20) and "Yintang" (EX-HN3) for 15 min, once daily, 6 days a week for 4 weeks for rats in the EA＋medication group. The Morris water maze (MWM) task (including place navigation tests and space exploration trials) was used to assess the mouse's learning-memory ability. Histopathological changes of hippocampus tissue were observed by H．E. staining. The expression levels of matrix metalloprotein 9 (MMP-9), low density lipoprotein receptor-related protein-1 (LRP-1), P-glycoprotein (Pgp, an important drug transporter responsible for multidrug resistance), Claudin-5 (a component of tight junction strands that serves as a physical barrier to prevent solutes and water from passing freely through the paracellular space between epithelial or endothelial cell sheets of blood-brain barrier, BBB) and Aß mRNAs of the hippocampus tissue were detected by quantitative real-time PCR. RESULTS: Compared with the control group, the average escape latency of place navigation tests, and the expression levels of MMP-9 and Aß mRNAs were significantly increased (P<0.01), and the number of platform quadrant-crossing times of space exploration trials, and the expression levels of LRP-1, Pgp and Claudin-5 mRNAs considerably decreased in the model group (P<0.01). After the intervention, the learning-memory ability was significantly improved in the medication and EA＋medication groups (P<0.01，P<0.05), the expression levels of Aß mRNAs in the medication and EA＋medication groups and MMP-9 mRNA in the EA＋medication group were obviously down-regulated (P<0.01), and those of LRP-1 and Pgp mRNAs in the medication and EA＋medication groups and Claudin-5 mRNA in the EA＋medication group were remarkably up-regulated (P<0.05, P<0.01). The therapeutic effect of EA＋medication was apparently superior to that of simple medication in shortening the escape latency (P<0.05，P<0.01) and in down-regulating the expression of MMP-9 and Aß mRNAs(P<0.01), and in increasing the number of platform quadrant-crossing times(P<0.01), and expression levels of LRP-1, Pgp and Claudin-5 mRNAs (P<0.01). H．E. staining showed scatted and loose arrangement of neurons in the hippocampus, with reduction of number of cell layers and unclear nucleoli, which was relatively milder in the medication and EA＋medication groups. CONCLUSION: EA can enhance the effect of Donepezil in improving learning-memory ability in AD mice possibly by regulating expression of MMP-9, LRP-1, Pgp and Claudin-5 mRNAs and strengthening the effect of Donepezil in transporting Aß via BBB.

[Effects of electroacupuncture on proangiogenesis process and protein turnover in a mouse model of sarcopenia].

OBJECTIVE: To observe the effect of electroacupuncture(EA) on proangiogenesis process and protein turn-over in a mouse model of sarcopenia, so as to explore its potential molecular mechanism anti-aging. METHODS: Fourteen 30-week-old male SAMP8 mice were randomly divided into a model group (n=7) and an EA group (n=7). Seven anti-rapidly aging SAMR1 mice of the same age were used as the control group (n=7). EA (1 mA, 4 Hz) was applied to bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 20 minutes each time once a day, 6 times a week for 4 weeks. The exhausted running platform was used to test the sports function. Gastrocnemius muscle mass and relative ratio of gastrocnemius muscle mass to body mass were measured. HE staining and transmission electron microscope were used to observe the morphology, and the cross-sectional area of gastrocnemius muscle was calculated. Relative protein expressions of protein kinase B (AKT) , phosphorylated (p) -AKT, mammalian target of rapamycin (mTOR) , p-mTOR, p70 ribosomal protein S6 kinase (p70S6K) , p-p70S6K,hypoxia inducible factor-1α (HIF-1α) and relative mRNA expressions of HIF-1α, vascular endothelial growth factor A (VEGF-A) , muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx) were detected by Western blot and real-time fluorescence quantitative PCR, seperatively. RESULTS: Compared with the control group, the running time and distance, body mass and gastrocnemius mass, and the ratio of gastrocnemius mass to body mass decreased(P<0.01, P<0.05), cross-sectional area of gastrocnemius, related protein expression of p-AKT,p-mTOR, p-p70S6K and HIF-1α, mRNA expression of HIF-1α and VEGF-A decreased (P<0.01), while mRNA expression of MuRF1 and MAFbx increased (P<0.01) in the model group. Following EA intervention, the running time and distance, body mass and gastrocnemius mass and the ratio of gastrocnemius mass to body mass increased (P<0.05), cross-sectional area of gastrocnemius, related protein expression of p-AKT,p-mTOR, p-p70S6K and HIF-1α, mRNA expression of HIF-1α and VEGF-A were significantly up-regulated (P<0.01), mRNA expression of MuRF1 and MAFbx down-regulated (P<0.01, P<0.05) in the EA group compared with the model group. CONCLUSION: EA may delay the aging muscle atrophy in mice by regulating the gastrocnemius muscle's proangiogenesis process and protein turnover.

Climate warming and cyanobacteria blooms: Looks at their relationships from a new perspective.

Climate warming and eutrophication are regarded as two important contributors to the occurrence of cyanobacteria blooms in aquatic ecosystems. However, the feedback of cyanobacteria blooms to climate warming and eutrophication is not fully clear. In this study, a microcosm system was established to simulate the decomposition processes of cyanobacteria blooms. It was observed that a large amount of nitrogen and phosphorus was released into the overlying water, and the concentrations of nitrogen and phosphorus were increased with the amount of added cyanobacteria bloom biomass addition. Subsequently, these released nutrients became available for primary production and intensified the eutrophic state of freshwater lakes. During the decomposition of cyanobacteria blooms, the microenvironment acquired low DO, low pH, and reductive conditions. Together with abundant organic matter in the water column and sediment, a large amount of CH4 and CO2 produced through organic matter mineralization, in which CH4 was the dominant fraction, occupied 50%-92% in mass of emitted carbon. Furthermore, a certain amount of N2O, probably underestimated, was produced with a strong greenhouse effect, even though its magnitude was small. These observations clarify that the feedbacks among cyanobacteria blooms formation and climate warming as well as the eutrophication of freshwater lakes are not unidirectional, but bidirectional. Given that climate warming enhanced the occurrence of cyanobacteria blooms, it was proposed that there are two vicious loops between cyanobacteria blooms, lake eutrophication and climate warming, which should be considered in the future management of aquatic ecosystems.