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.

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.

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.

[Effects of electroacupuncture on autophagy factors and liver lipid metabolism in rapidly aging mice].

Objective: To study the effects of electroacupuncture on the expressions of autophagy-related factors LC3-Ⅱ, Beclin1, Atg7, and P62 in the liver of rapidly aging (senescence accelerated mouse/prone8,SAMP8) mice, and to explore the mechanisms of electroacupuncture to improve liver lipid metabolism in mice. Methods: Thirty-week-old male SAMP8 mice were randomly divided into model group, drug group, and electroacupuncture group, with 7 mice in each group. Seven anti-rapid aging SAMR1 mice of the same age were used as the control group. The animals in the control group and the model group were bred routinely for 2 weeks without any intervention; the drug group was treated with intraperitoneal injection of rapamycin at the dose of 10 mg·kg-1·d-1, once a day, 6 consecutive days a week; the electroacupuncture group was given "Shenshu" and "Taichong" Electroacupuncture at point(15 minutes a day, 6 consecutive days a week). The serum lipid metabolism and liver lipid deposition of mice were detected, the distribution of liver autophagy body, the protein and mRNA expressions of liver LC3 - Ⅱ, Beclin1, Atg7 and P62 were determined. Results: Compared with the control group, the total cholesterol (TC), triglycerides (TG), and low density lipoprotein (LDL) of the model group were increased significantly(P＜0.01). In the model group, lipid droplet deposition was obvious, autophagosomes were decreased, the protein and mRNA expression levels of autophagy- related factors LC3-Ⅱ, Beclin1 and Atg7 were decreased significantly (P＜0.01), while the protein and mRNA expressions of P62 were increased significantly (P＜0.01). Compared with the model group, the serum contents of TG, TC, and LDL of the mice in the electroacupuncture group and the drug group were decreased significantly (P＜0.01), lipid droplet deposition was reduced, autophagosomes were increased, the protein and mRNA expression levels of LC3 -Ⅱ, Beclin1 and Atg7 were increased significantly(P＜0.01), and the protein and mRNA expression levels of P62 were decreased significantly(P＜0.01). The protein and mRNA expression levels of Beclin1 and Atg7 in the liver of the electroacupuncture group were not significantly different from the drug group (P＞0.05). Conclusion: Electroacupuncture can alleviate liver lipid metabolism disorders, which may be related to the regulation of the expressions of liver autophagy related factors LC3-Ⅱ, Beclin1, Atg7, and P62, thereby promoting liver autophagy in SAMP8 mice.

[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.

[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.