Effects of electroacupuncture on Sirt3/NLRP3/GSDMD signaling pathway in the substantia nigra of midbrain of rats with Parkinson's disease. / ç”µé’ˆå¯¹å¸•é‡‘æ£®ç— å¤§é¼ ä¸­è„‘é»‘è´¨Sirt3/NLRP3/GSDMD信号通路的影响.

OBJECTIVES: To observe the effects on tyrosine hydroxylase (TH), α-synaptic nucleoprotein (α-syn), sirtuin 3 (Sirt3), NOD-like receptor 3 (NLRP3) and gasdermin-D (GSDMD) in the substantia nigra of midbrain after electroacupuncture (EA) at "Fengfu"(GV16), "Taichong" (LR3) and "Zusanli" (ST36) in rats of Parkinson's disease (PD), so as to explore the mechanism of EA in treatment of PD. METHODS: SD rats were randomly divided into control, model and EA groups, with 10 rats in each group. The PD model was established by injecting rotenone into the neck and back, lasting 28 days. In the EA group, EA was applied to GV16, LR3 and ST36, 30 min each time, once daily, consecutively for 28 days. The open-field test was adopted to detect the total distance of autonomic movement of rats, and the pole climbing test was used to detect the body coordination ability of rats. In the substania nigra of midbrain, the positive expression of TH was determined using immunohistochemistry, the mRNA expression levels of α - syn, Sirt3, NLRP3 and GSDMD were detected by quantitative real-time fluorescence PCR, and the protein expression levels of NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and cysteinyl aspartate specific proteinase (Caspase)-1 were detected by Western blot. RESULTS: Compared with the control group, the total distance of autonomous movement was decreased (P<0.01) in the model group, and the score of pole climbing experiment was increased (P<0.01)；in the midbrain substantia nigra the positive expression of TH was decreased (P<0.01)；the mRNA expression level of Sirt3 was decreased (P<0.01), and those of α-syn, NLRP3 and GSDMD were increased (P<0.01)；while the protein expression levels of NLRP3, ASC and Caspase-1 were increased (P<0.01). When compared with the model group, the total distance of autonomous movement in open field experiment was increased (P<0.01) in the EA group and the score of pole climbing experiment was lower (P<0.05)；in the midbrain substantia nigra the positive expression of TH was increased (P<0.01)；the mRNA expression level of Sirt3 in the midbrain substantia nigra was increased (P<0.01), and those of α-syn, NLRP3 and GSDMD were reduced (P<0.01)；while the protein expression levels of NLRP3, ASC and Caspase-1 decreased (P<0.01, P<0.05). CONCLUSIONS: EA at "GV16" "LR3" and "ST36" can repair the neuronal injury, clear the abnormal accumulation of α-syn in the substania nigra of midbrain, and ameliorate mitochondrial damage in PD rats, which may be obtained by regulating Sirt3/NLRP3/GSDMD signaling pathway, so as to delay the occurrence and development of Parkinson's disease.

Effects of electroacupuncture on mitophagy mediated by SIRT3/PINK1/Parkin pathway in Parkinson's disease mice. / ç”µé’ˆå¯¹å¸•é‡‘æ£®ç— å°é¼ SIRT3/PINK1/Parkin通路介导的线粒体自噬的影响.

OBJECTIVES: To observe the effects of electroacupuncture (EA) at "Fengfu"(GV16), "Taichong"(LR3), and "Zusanli"(ST36) on mitophagy mediated by silencing regulatory protein 3 (SIRT3)/ PTEN induced putative kinase 1 (PINK1)/PARK2 gene coding protein (Parkin) in the midbrain substantia nigra of Parkinson's disease (PD) mice, and to explore the potential mechanisms of EA in treating PD. METHODS: C57BL/6 mice were randomly divided into the control, model, EA, and sham EA groups, with 12 mice in each group. The PD mouse model was established by intraperitoneal injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). The EA group received EA stimulation at GV16, LR3 and ST36, while the sham EA group received shallow needling 1 mm away from the above acupoints without electrical stimulation. The motor ability of mice in each group was evaluated using an open field experiment. Immunohistochemistry was used to detect the expression of tyrosine hydroxylase (TH) and α-synuclein (α-syn) in the substantia nigra of mice. The ultrastructure of neurons in substantia nigra was observed by transmission electron microscope (TEM). Immunofluorescence was used to detect the expression of the autophagy marker autophagy-associated protein light chain 3 (LC3). The expression levels of TH, α-syn, SIRT3, PINK1, Parkin, P62, Beclin-1, LC3Ⅱ mRNA and protein were detected by PCR and Western blot. RESULTS: Compared with the control group, mice in the model group showed a decrease in the total exercise distance, time, movement speed and times of crossing central region (P<0.01)；the positive expressions of TH and LC3 were decreased (P<0.01), while the positive expression of α-syn increased (P<0.01), accompanied by mitochondrial swelling, mitochondrial cristae fragmentation and decrease, and decreased lysosome count；the expression levels of TH, SIRT3, PINK1, Parkin, Beclin-1, and LC3Ⅱ mRNA and protein in the midbrain substantia nigra were decreased (P<0.01), while the expression levels of α-syn and P62 mRNA and protein were increased (P<0.01, P<0.05). Compared with the model group, the mice in EA group showed a significant increase in the total exercise distance, time, movement speed and times of crossing central region (P<0.01, P<0.05)；the positive expressions of TH and LC3 were increased (P<0.01, P<0.05), while the positive expression of α-syn was decreased (P<0.01), accompanied by an increase in mitochondrial count, appearance of autophagic va-cuoles, and a decrease in swelling, the expression levels of TH, SIRT3, PINK1, Parkin, Beclin-1 and LC3Ⅱ mRNA and protein in the midbrain substantia nigra were increased (P<0.01, P<0.05), while the mRNA and protein expression levels of α-syn and P62 were decreased (P<0.01)；the sham EA group showed an increase in the total exercise distance and time(P<0.05), with an increase in the positive expression of TH (P<0.05) and a decrease in the positive expression of α-syn (P<0.05)；some mitochondria exhibited swelling, and no autophagic vacuoles were observed；the protein expression levels of TH, SIRT3, Parkin and LC3Ⅱ were increased (P<0.01, P<0.05), and the expression levels of P62 mRNA, α-syn mRNA and protein were decreased (P<0.01, P<0.05), and LC3Ⅱ mRNA expression was increased (P<0.05). In comparison to the sham EA group, the EA group showed an extension in the total exercise time (P<0.01), the positive expression and mRNA expression levels of α-syn were decreased (P<0.01, P<0.05), while the expression levels of TH, SIRT3, PINK1, Parkin mRNA and SIRT3 protein were increased (P<0.05). CONCLUSIONS: EA at GV16, LR3, and ST36 can exert neuroprotective function and improve the motor ability of PD mice by activating the SIRT3/PINK1/Parkin pathway to enhance the expression of TH and reduce α-syn aggregation in the substantia nigra of PD mice.

Effects of electroacupuncture on the intestinal thioredoxin interaction protein/Nod-like receptor 3 signaling pathway in mice with Parkinson's disease. / ç”µé’ˆå¯¹å¸•é‡‘æ£®ç— å°é¼ è‚ é“ç¡«æ°§è¿˜è›‹ç™½äº’ä½œè›‹ç™½/NODæ ·å—ä½“3信号通路的影响.

OBJECTIVES: To observe the effects of electroacupuncture (EA) at "Fengfu" (GV16), "Taichong" (LR3) and "Zusanli" (ST36) on α-synuclein (α-syn), Occludin, Claudin-1, thioredoxin interaction protein (TXNIP) and Nod-like receptor 3 (NLRP3) in Parkinson's disease (PD) mice, so as to investigate the mechanisms of EA on intestinal barrier function and inflammation in PD mice. METHODS: Thirty six C57BL/6 mice were randomly divided into control, model and EA groups, with 12 mice in each group. PD mice model was induced by rotenone intragastric administration for 28 days. Mice in the EA group were treated with EA (2 Hz, 1 mA) at GV16, LR3 and ST36 for 30 min, once a day for 14 days. The behavioral scores were observed. The total distance of autonomic movement was measured by open field test. The expression level of α-syn in substantia nigra and colon tissue was determined by immunohistochemistry. The colonic morphology and goblet cell distribution were observed by Alcian blue staining. The expression levels of Occludin, Claudin-1, TXNIP and NLRP3 mRNA in colon tissue were detected by real-time fluorescence quantitative PCR. RESULTS: Compared with the control group, the behavioral scores of rats were increased (P<0.01)；the total distance of autonomous movement was decreased (P<0.01)；the positive expression level of α-syn in the substantia nigra and colon was increased (P<0.01)；the goblet cells and crypts in colon tissue were reduced, and the muscular layer was thinner；the expression levels of Occludin and Claudin-1 mRNAs in colon tissue were decreased (P<0.01) while TXNIP and NLRP3 mRNAs were increased (P<0.01) in the model group. Compared with the model group, the surface villi of colon tissue was more complete, the goblet cells and crypts were increased, and the muscular layer was thickened；the other indexes were reversed (P<0.01, P<0.05) in the EA group. CONCLUSIONS: EA at GV16, LR3 and ST36 can reduce the abnormal accumulation of α-syn in the substania nigra and colon tissue of PD mice, alleviate the damage of intestinal barrier, regulate TXNIP/NLRP3 signaling pathway, so as to delay the occurrence and development of PD.

Targets and mechanisms of Alpinia oxyphylla Miquel fruits in treating neurodegenerative dementia.

The dried and ripe fruits of Alpinia oxyphylla and ripe fruits of Alpinia oxyphylla Miquel (AO) have the effects of tonifying kidney-essence and nourishing intelligence and thus have been widely used in treating dementia. Alzheimer's disease (AD) is a typical form of neurodegenerative dementia with kidney-essence deficiency in Traditional Chinese Medicine (TCM). So far, there is a lack of systematic studies on the biological basis of tonifying kidney-essence and nourishing intelligence and the corresponding phytochemicals. In this study, we investigated the targets of AO in tonifying kidney-essence and nourishing intelligence based on the key pathophysiological processes of neurodegenerative dementia. According to ultra-high-performance liquid chromatography with triple quadrupole mass spectrometry data and Lipinski's rule of five, 49 bioactive phytochemicals from AO were identified, and 26 of them were found to target 168 key molecules in the treatment of neurodegenerative dementia. Nine phytochemicals of AO were shown to target acetylcholinesterase (ACHE), and 19 phytochemicals were shown to target butyrylcholinesterase (BCHE). A database of neurodegenerative dementia with kidney-essence deficiency involving 731 genes was constructed. Furthermore, yakuchinone B, 5-hydroxy-1,7-bis (4-hydroxy-3-methoxyphenyl) heptan-3-one (5-HYD), oxyhylladiketone, oxyphyllacinol, butyl-ß-D-fructopyranoside, dibutyl phthalate, chrysin, yakuchinone A, rhamnetin, and rhamnocitrin were identified as the key phytochemicals from AO that regulate the pathogenesis of neurodegenerative dementia in a multitargeted manner. The approach of studying the pharmacological mechanism underlying the effects of medicinal plants and the biological basis of TCM syndrome may be helpful in studying the translation of TCM.

[Mechanism of electroacupuncture against intestinal inflammation in mice with Parkinson's di-sease by NF-κB/IL-6 pathway].

OBJECTIVE: To observe the effects of electroacupuncture (EA) at "Fengfu"(GV16), "Taichong"(LR3), and "Zusanli"(ST36) on the tyrosine hydroxylase (TH), nuclear factor-kappa B (NF-κB), interleukin (IL)-6, and zonula occludens-1 (ZO-1) in mice with Parkinson's disease (PD), and to explore the underlying mechanism of EA in the treatment of PD. METHODS: C57BL/6 mice were randomly divided into control, model, and EA groups, with 10 mice in each group. The PD model was induced by rotenone (i.g.) in mice for 28 d. EA was applied to GV16, LR3 and ST36 of mice in the EA group for 30 min, once daily for 14 d. The behavioral changes of mice in each group before and after treatment were observed and scored. The total distance traveled autonomously of mice was detected in the open field test. TH expression in the substantia nigra (SN) was measured by immunohistochemistry, and the changes in colon tissue structure were observed by HE staining. The intestinal mRNA expression of ZO-1, NF-κB, and IL-6 was detected by quantitative real-time PCR, and the colon tissue protein expression of NF-κB was detected by Western blot. The colon tissue concentration of IL-6 was detected by ELISA. RESULTS: Compared with the control group, the pre- and post-treatment behavioral scores of the model group and the pre-treatment behavioral score of the EA group were increased (P<0.01). Compared with the model group, the EA group showed reduced behavioral score after treatment (P<0.01). Compared with the control group, the model group showed reduced goblet cells and crypts and thinner muscle layer in the intestinal specimens of mice. Compared with the model group, the EA group showed intact surface villi, increased goblet cells and crypts, and thickened muscle layer. Compared with the control group, the model group exhibited reduced total distance traveled in the open field test, TH expression in SN, and ZO-1 mRNA expression (P<0.01), and elevated mRNA and protein expression of NF-κB and IL-6, and the ratio of p-NF-κB/NF-κB in the intestinal tract (P<0.01). Compared with the model group, the EA group displayed increased total distance traveled, TH expression in SN, and ZO-1 mRNA expression (P<0.01), and declining mRNA and protein expression of NF-κB and IL-6, and the ratio of p-NF-κB/NF-κB in the colon tissue (P<0.01, P<0.05). CONCLUSION: EA at GV16, LR3, and ST36 can regulate the expression of NF-κB/IL-6, inhibit the transmission of the colon tissue inflammatory response, repair the intestinal barrier function, and potentiate the TH activity, thereby improving the behavioral performance of PD mice.

[Study on proteins in Guangdilong by nano LC/orbitrap fusion lumos HR-MS].

In the present study,fresh Guangdilong( GD),originating from Pheretima aspergillum,was taken as the object. The total proteins from GD were firstly separated by SDS-PAGE according to their molecular weights and in-gel digestion was then performed.After that,the peptides were analyzed by nano LC/orbitrap fusion lumos high resolution mass spectrometry( nano LC/orbitrap fusion lumos HR-MS). Protein identification was implemented by comparison with Annelida. fasta database using Proteome Discoverer software.As a result,386 proteins were tentatively identified,including chain F,globin B chain,glyceraldehyde-3-phosphate dehydrogenase,fibrinolytic protein,and so on. Most of the proteins took part in cell structure and energy metabolism,and fibrinolytic protein and lombricine kinase might be related to fibrinolytic activity. Protein classification based on gene ontology was carried out using PANTHER and KEGG for metabolic pathway enrichment. The results indicated that these proteins were related to diverse signal transduction pathways,including metabolic pathways,central carbon metabolism,biosynthesis of amino acids,ribosome,glycolysis,citrate cycle( TCA cycle),and so on. This study would lay the foundation for the further research on the proteins in GD and also their functions.

Icariin protects cardiomyocytes against ischaemia/reperfusion injury by attenuating sirtuin 1-dependent mitochondrial oxidative damage.

BACKGROUND AND PURPOSE: Icariin, a major active ingredient in traditional Chinese medicines, is attracting increasing attention because of its unique pharmacological effects against ischaemic heart disease. The histone deacetylase, sirtuin-1, plays a protective role in ischaemia/reperfusion (I/R) injury, and this study was designed to investigate the protective role of icariin in models of cardiac I/R injury and to elucidate the potential involvement of sirtuin-1. EXPERIMENTAL APPROACH: I/R injury was simulated in vivo (mouse hearts), ex vivo (isolated rat hearts) and in vitro (neonatal rat cardiomyocytes and H9c2 cells). Prior to I/R injury, animals or cells were exposed to icariin, with or without inhibitors of sirtuin-1 (sirtinol and SIRT1 siRNA). KEY RESULTS: In vivo and in vitro, icariin given before I/R significantly improved post-I/R heart contraction and limited the infarct size and leakage of creatine kinase-MB and LDH from the damaged myocardium. Icariin also attenuated I/R-induced mitochondrial oxidative damage, decreasing malondialdehyde content and increasing superoxide dismutase activity and expression of Mn-superoxide dismutase. Icariin significantly improved mitochondrial membrane homeostasis by increasing mitochondrial membrane potential and cytochrome C stabilization, which further inhibited cell apoptosis. Sirtuin-1 was significantly up-regulated in hearts treated with icariin, whereas Ac-FOXO1 was simultaneously down-regulated. Importantly, sirtinol and SIRT1 siRNA either blocked icariin-induced cardioprotection or disrupted icariin-mediated mitochondrial homeostasis. CONCLUSIONS AND IMPLICATIONS: Pretreatment with icariin protected cardiomyocytes from I/R-induced oxidative stress through activation of sirtuin-1 /FOXO1 signalling.

[Molecular mechanism of Bupleuri Radix and Scutellariae Radix drug pair for depression based on integrative pharmacology platform of traditional Chinese medicine].

Xiaochaihu decoction is a classic prescription of traditional Chinese medicine. Modern research has proved its anti-depression effect. However, its pharmacological mechanism for anti-depression effect is difficult to be unveiled because of the complexity of compound Chinese medicines. Bupleuri Radix and Scutellariae Radix is the core drug pair of Xiaochaihu decoction. In this research, Bupleuri Radix and Scutellariae Radix were analyzed by the integrative pharmacology platform to study its molecular mechanism for anti-depression. One hundred and sixteen active ingredients were predicted, 62 for Bupleuri Radix, mainly including saikosaponins, acids, alcohols, and 54 for Scutellariae Radix, mainly including flavonoids and glycosides. Its anti-depression effect was relevant to 118 core targets, including 22 known disease targets, such as serotonin receptor(HTR2C), activating transcription factor(ATF1, ATF2), δ opioid receptor(OPRD1), µ opioid receptor (OPRM1), κ opioid receptor(OPRK1), inositol monophosphatase(IMPA1), Toll-like receptor 4 (TLR4), histamine H1 receptor(HRH1), neurotrophic factor tyrosine kinase receptor1 (NTRK1), Glycogen synthetase kinase 3ß(GSK3ß), etc. The antidepressant effect involved positive regulation of transcription from RNA polymerase Ⅱ promoter, transcription factor binding, cytosol, transcriptional regulation of DNA template, enzyme binding, endocrine system, nervous system, neurotrophin signaling pathway, cell growth and death, signal transduction, thyroid hormone signaling pathway and other related biological processes and metabolic pathways. This study provides a scientific evidence for further study of the anti-depression mechanism of this drug pair.

[Effect of Electroacupuncture Intervention on Expression of Cx 43 and Concentration of Glutamate in the Striatum of Rats with Parkinson's Disease].

OBJECTIVE: To observe the action of electroacupuncture (EA) intervention on the expression of gap junction protein connexin 43 (Cx 43) and content of glutamate (Glu) in the striatum in Parkinson's disease (PD) rats, so as to reveal its mechanism underlying improvement of PD. METHODS: Forty male SD rats were randomly divided into normal control, sham operation, model and EA groups (n = 10 in each group). The PD model was duplicated by microinjection of 6-hydroxyldopamine (6-OHDA, 15 µg/rat) into the right striatum of rats (AP: 1.0, 1.0; R: 3.0, 4.5; H: 4.5, 6.0), and for control, the same dose of normal saline was injected into the right striatum for rats in the sham operation group. EA (2 Hz, 1 mA) was applied to "Fengfu" (GV 16) "Taichong" (LR 3) for 30 min, once a day for 2 weeks. The PD rats' rotational behavior changes (the numbers of rotations in 30 min) were detected following subcutaneous injection of apomorphine (0.5 mg/kg). The Glu concentration and the expression of Cx 43 in the striatum were detected by using high performance liquid chromatography (HPLC) and Western blot, respectively. RESULTS: No significant differences were found between the model group and EA group in the number of rotations before the treatment, between the control and sham operation groups in the levels of Glu content and Cx 43 protein expression in the striatum (P > 0.05). Compared with the control group, the Glu content and Cx 43 protein expression level were significantly increased in the model group (P < 0.01), while in comparison with the model group, the number of rotations was significantly reduced in the EA group (P < 0.05). Following EA intervention, both Glu content and Cx 43 expression were considerably down-regulated in the EA group compared with the model group (P < 0.05, P < 0.01). CONCLUSION: EA can improve PD rats' rotation behavior, which may be associated with its effects in down-regulating the level of Glu and Cx 43 protein expression in the striatum.

[Effect of Electroacupuncture on 26 S Proteasome and Nuclear Factor kappa B in Substantia Nigra of Rats with Rotenone-induced Parkinson's Disease].

OBJECTIVE: To explore the role of 26 S proteasome and nuclear factor kappa B (NFκB) in substantia nigra in the management of Parkinson's disease (PD) by acupuncture. METHODS: Forty-eight male SD rats were randomly divided into normal, sham operation, model and electroacupuncture (EA) groups (12 rats/group).. The PD model was established by 40-day consecutive subcutaneous injection of rotenone (1 mg/kg dissolved in dimethyl sulfoxide and normal saline) at the back shoulder. The rats in the sham operation group were treated by subcutaneous injection of dose of saline. "Fengfu" (GV 16) and "Taichong" (LR 3) acupoints were stimulated with EA at 2 Hz, 1 mA, 20 min of duration in each treatment, and daily for 28 consecutive days. The behavioral changes of rats in each group were measured and scored at 40th day and 68th day, respectively. Immunohistochemistry was used to determine the expression of tyrosine hydroxylase (TH) used to detect the expression of 26 S proteasome and NFκB and TH were measured by Western blot. RESULTS: In comparison with the normal and sham operation groups, the behavioral scores of rats in the model group were elevated, which were significantly decreased by EA intervention (P < 0.05). The expression of TH and 26 S proteasome decreased whereas the NFκB increased in the rats of model group (P < 0.05); and EA intervention reversed these changes (all P < 0.05). CONCLUSION: EA intervention can improve PD rats' behavioral changes, which is pobably related to its effects in reducing loss of TH-positive neurons, down-regulating NFκB protein expression, and up- regulating 26 S proteasome protein expression in the substantia nigra.

[Effects of Hemerocallis citrine baroni flavonids on CCl4-induced liver fibrosis of rats].

This study is designed to explore the possible effects of Hemerocallis citrina baroni flavonids (HCBF) on liver fibrosis induced by CCl4 in rats. The liver fibrosis model was induced by CCl4, and HCBF were administered by gastric perfusion at 25 and 50 mg x kg(-1) qd for 50 days, while the contents of alanine transaminase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), superoxide dismutase (SOD), maleic dialdehyde (MDA) and transforming growth factor-ß1 (TGF-ß1) were measured and the contents of PINP were measured in liver tissue, and the expression of TGF-ß1 were observed by immunohistochemisty and Western blot. The pathological changes of liver tissue were examined by HE. The results showed that HCBF (25, 50 mg x kg(-1)) improved the liver function significantly through reducing the level of ALT, AST, GGT and ALP (P < 0.05 or P < 0.01), and increasing the content of SOD (P < 0.01), while reducing the content of MDA (P < 0.05 or P < 0.01), the expression of TGF-ß1 (P < 0.05) and the content of PINP (P < 0.05). The results suggest that HCBF (25, 50 mg x kg(-1)) may inhibit the liver injury induced by CCl4 by decreasing the oxidative stress.

[Effect of flavonoids from Sophora flavescens in aging mice induced by D-galactos].

To investigate the effect of flavonoids from Sophora flavescens in aging mice induced by D-galactose and its mechanism. Totally 60 mice were randomly divided into six groups: the control group, the model group, the piracetam group (positive control group) and flavonoids from S. flavescens low, medium and high doses groups. Except for the control group, all of the rest groups were subcutaneously injected with D-galactose (160 mg x kg(-1)) for successively 30 days to establish the sub-acute senescent model. Meanwhile, flavonoids from S. flavescens low, medium and high doses groups were respectively administered with 150, 300 and 600 mg xkg-('1)of flavonoids from S. flavescens for 30 days. The learning and memory abilities of mice were determined by avoiding darkness ex-eriment and jumping stair experiment. The contents of malondialdehyde (MDA) tumor necrosis factor-aα NF-aα the activities of superoxide dismutase (SOD) monoamine oxidase-B (MAO-B) Na'(+)K'(+)-ATPase and Ca2(+ )-ATPase in the brain of mice were deter-ined respectively after the behavioral experiments. The activity of lactic dehydrogenase ( DH) in blood serum was also determined and analyzed by microscope after HE staining to observe the changes in hippocampal organizational structure. Compared with the model group, flavonoids from S. favescens medium and high doses groups showed significantly increases in the latency of avoiding darkness and jumping stair experiments; flavonoids from S. fllvescens low, medium and high doses groups and the piracetam group showed de-reases in the numbers of errors in avoiding darkness experiment; the flavonoids from S. flavescens high dose group and the piracetam group showed reduction- n the number of errors in jumping stair experiment (P <0 . 5 or P <0 . 1). Flavonoids from S. flavescens me-ium and high doses groups and the piracetam group showed improvements in the activities of SOD, Na'(+)K'(+)ATPase in the brain of mice and declines in the contents of MDA and TNF-aα the activity of MAO-B in the brain of mice, the activity of LDH in blood serum (P <0 . 5 or P <0 . 1). Flavonoids from S. flavescens low, medium and high doses groups and the piracetam group also showed im-rovement in the activity of Ca2(+ )ATPase, with statistical difference from the model group (P <0 . 5 or P <0 . 1). The pathological result showed decreases in the number of cells of hippocampal dentate gyrus area, sparse cell arrangement, incomplete cellular mor-hology, scarce cytoplasm, blurred boundary between nucleus and cytoplasm, nuclei anachromasis, irregular pyknosis and unconspicu-us nucleoli in the model group. Compared with the model group, flavonoids from S. flavescens low, medium and high doses groups and the piracetam group showed improvements in hippocampal organization tissues. Flavonoids from S. favescens can improve the learning and memory ability of senescent mice induced by D-galactose. Its mechanism may be correlated with the enhancement of anti-oxidative actions by lowering TNF-aαcontent, which results in the stability of cell membrane and the reduction in MAO-B activity.

[Effect of electroacupuncture intervention on ERK 1/2 signaling and TNF-α and IL-1ß protein levels in the substantia Nigra in rats with Parkinson's Disease].

OBJECTIVE: To observe the effect of electroacupuncture(EA) on phosphalized extracellular regulated protein kinases(p-ERK) 1/2 signaling pathway and tumor necrosis factor (TNF)-α and IL-1ß protein levels in the substantia nigra (SN) in rats with Parkinson's disease (PD), so as to explore its mechanism underlying improvement of PD. METHODS: 32 male SD rats were randomly and averagely divided into normal control group, sham-operation group, model group and EA group. PD model was established by intra-dermal-injection of rotenone(1 mg/kg, dissolved in DMSO and saline, concentration: 0. 25 mg/mL) at the nape, once daily for 14 days. EA (2 Hz, 2 mA) was applied to "Fengfu"(GV 16)and "Taichong"(LR 3) for 20 min, once daily for 14 days. For sham-operation group, subcutaneous injection of the same dose of DMSO and saline was given in the same way. The expression levels of tyrosine hydroxylase (TH), p-ERK 1/2, inflammatory cytokines TNF-α and IL-1P proteins of the SN tissue were detected using Western blot. The rat's horizontal and vertical movement ability was assessed using open-field tests. RESULTS: The horizontal and vertical movement scores were significantly lower in the model group than in the normal control group, and markedly higher in the EA group than in the model group (P<0. 05). Compared with the normal control group, the expression level of TH protein in the SN was significantly reduced in the model group( P<0. 05 ),while the expression levels of p-ERK 1/2, TNF-α and IL-1ß proteins were significantly increased in the model group(P<0. 05). After EA intervention, the expression level of TH protein was significantly increased(P<0.05),and those of p-ERK 1/2, TNF-α and IL-1ß proteins were significantly down-regulated(P<0. 05) in the EA group compared to the model group. CONCLUSION: EA therapy may improve PD rats' movement ability, which may be associated with its effects in down-regulating the expression levels of p-ERK 1/2, TNF-α and IL-1ß proteins and up-regulating the expression of TH protein in the SN.

[Impacts of electroacupuncture on ubiquitin-proteasome system in rats with Parkinson's disease].

OBJECTIVE: To explore action mechanism of electroacupuncture (EA) on treatment and prevention of Parkinson's disease (PD). METHODS: Fifty clean-grade SD rats were randomly divided into a normal group, a control group, a model group, a pretreatment group and a treatment group, ten rats in each one. The PD model was established by subcutaneous injection of rotenone in neck-back skin (2 mg/kg, dissolved in sun-flower oil, 2 mg/mL in density). The equal-volume sun-flower oil that didn't include rotenone was applied in the control group at the same area as the model group. EA was applied in the treatment group at "Fengfu" (GV 16) and "Taichong" (LR 3) with interrupted wave, 2 Hz in frequency, 1 mA in density, for 20 min. The treatment was given once day for conti-nuous 28 days. Rats in the pretreatment group received the same EA as the treatment group for 7 days, and then put into model establishment. After the model establishment, the rats received no treatment and were sacrificed after 28 days. No EA was given in the normal group, model group and control group. The ethology changes were observed and scored. The expression of Parkin, ubiquitin C terminal hydrolase-L1 (UCH-L1) and ubiquitin activating enzyme-1 (UBE1) in substantia nigra was tested by Western-blot method. The positive cell numbers of alpha-synuclein, ubiquitin (UB) and tyrosine hydroxylase (TH) in substantia nigra was tested by immunohistochemical method. RESULTS: There were abnormal ethology manifestation such as yellow and coarse hair, arched back, weaken behavior of resisting arrest and slow movement, which was more relieved in the treatment group and pretreatment group. Compared with normal group and control group, the expression of Parkin, UCH-L1, UBE1, UB, TH in the model group was obviously decreased while alpha-synuclein was obviously increased (all P<0.01). After EA or pretreatment, the expression of Parkin, UCH-L1, UBE1, UB, TH in the treatment group and pretreatment group was higher than that in the model group while expression of alpha-synuclein in the treatment group and pretreatment group was lower than that in the model group (all P<0.01). CONCLUSION: EA or pretreatment could not only have protective effect for rats with PD, but also increase function of ubiquitin-proteasome system, indicating action mechanism of EA on treatment and prevention of PD may be related with ubiquitin-proteasome system.

[Effect of electroacupuncture stimulation of "Fengfu" (GV 16) and "Taichong" (LR 3) on expression of COX-2 and tyrosine hydroxylase in Substantia Nigra in rats with Parkinson's disease].

OBJECTIVE: To observe the influence of electroacupuncture (EA) therapy on the expression of tyrosine hydroxylase (TH) and cyclooxygenase-2 (COX-2) proteins of Substantia Nigra cells(SNc) in the rotenone-induced Parkinson's disease(PD) rats, so as to explore the mechanism of EA underlying improvement of PD. METHODS: Forty rats were randomly divided into normal, sham-operation (sham), model and EA groups (n = 10/group). The PD model was established by successive subcutaneous injection of rotenone (highly selective lesions of nigrostriatal dopaminergic neurons) for 28 days. EA (2 Hz, 1 mA) was applied to bilateral "Fengfu" (GV 16) and "Taichong" (LR 3) for 20 min, once daily for 14 days. The expression levels of TH and COX-2 proteins in the Substantia Nigra of midbrain were detected with Western blotting. RESULTS: Compared with the normal group, the expression level of TH protein in the model group was significantly decreased (P < 0.01), and that of COX-2 protein in the model group was significantly increased (P < 0.01). After the EA treatment, the expression level of TH in the EA group was obviously upregulated (P < 0.01), and that of COX-2 protein in the EA group was considerably down-regulated (P < 0.01). No significant differences were found between the normal and sham groups in the expression levels of TH and COX-2 proteins (P > 0.05). CONCLUSION: EA therapy can decrease inflammation mediator COX-2 protein expression and upregulate TH protein expression in the Substantia Nigra of midbrain in PD rats, which may contribute to its effect in relieving PD in clinic.

[Influence of electroacupuncture on p38-mitogen activated protein kinase in substantia nigra cells of rats with Parkinson disease model].

OBJECTIVE: To explore the role of inflammatory reaction mediated by p38-mitogen activated protein kinase (p38-MAPK) signal path on prevention and treatment of Parkinson disease (PD) model rats by electroacupuncture (EA). METHODS: Thirty-two healthy male SD rats were randomly divided into a normal group, a sham operation group, a model group and an EA group, eight rats in each one. The PD model was established in the model group and EA group by subcutaneous injection of rotenone in skin-back area (2 mg/kg, dissolved in sunflower oil, 2 mg/mL in density), while the injection of sunflower oil emulsion without rotenone at the same point and quantity as the model group was applied in the sham operation group. The normal group was not given any intervention. The EA treatment (continuous wave, 2 Hz in frequency, 1 mA in intensity, 20 min) was applied at "Fengfu" (GV 16) and "Taichong" (LR 3) in the EA group, once a day for continuously 14 days. No treatment was given in the other groups. The expression of tyrosine hydroxylase (TH), phosphorylated p38-MAPK, cyclooxygenase-2 (COX-2) in the substantia nigra were detected with immunohistochemical method. RESULTS: There was typical PD ethology change in the model group. Compared with the normal group and sham operation group, the expression of TH positive neuron in the substantia nigra in the model group was significantly decreased, while the expression of phosphorylated p38-MAPK and COX-2 were significantly increased (all P < 0.01). Compared with the model group, the expression of TH positive neuron in the EA group was apparently increased, while the expression of phosphorylated p38-MAPK and COX-2 were significantly decreased (all P < 0.01). CONCLUSION: The EA therapy could obviously reduce the expression of inflammation mediator COX-2, inhibit the phosphorylation of p38-MAPK, reduce the damage of dopaminergic neurons in the rats with PD, and this effect may be related with the impact of p38-MAPK signal path

[Effects of electroacupuncture on the expression of GDNF and Ret in Parkinson's disease model rats].

OBJECTIVE: To explore the mechanism of electroacupuncture therapy on Parkinson's disease (PD). METHODS: Fifty Wistar rats were randomly divided into a normal group, a sham-operation group, a model group, a Fengfu-Taichong group and a Shuanggu Yitong group. PD model was duplicated by microinjection of 6-Hydroxyl-Dopamine into right corpora striata, and by microinjection of normal saline in sham-operation group. Rats in normal group, sham-operation group and model group were not treated. In Fengfu-Taichong group, the rats were treated by electroacupuncture at "Fengfu" (GV 16) and "Taichong" (LR 3) on the basis of the PD model, and by electroacupuncture at "Fengfu" (GV 16), "Taichong" (LR 3), "Guanyuan" (CV 4) and "Zusanli" (ST 36) in Shuanggu Yitong group, once daily for 2 weeks. GDNF and Ret expression were detected by immunohistochemistry and western blotting, respectively. RESULTS: The number of GDNF positive cells and the content of Ret receptor increased significantly in the two electroacupuncture groups compared with those in the other groups (all P < 0.01), and the expression of GDNF increased significantly in Shuanggu Yitong group compared with that in Fengfu-Taichong group (P < 0.01). CONCLUSION: Electroacupuncture can not only increase the expression of GDNF, but also enhance its effect. "Shuanggu Yitong" method is better than simple acupuncture at "Fengfu" (GV 16) and "Taichong" (LR 3) in increasing expression of GDNF.

[Effect of electroacupuncture on morphological changes and apoptosis of substantia nigra cells in Parkinson's disease rats].

OBJECTIVE: To observe the influence of electroacupuncture (EA) of "Fengfu" (GV16) and "Taichong" (LR3) on morphological changes and apoptotic percentage of substantia nigra cells so as to explore its underlying mechanism in relieving Parkinson's disease. METHODS: Forty Wistar rats were randomly divided into normal control, sham-operation (sham), model and EA groups, with 10 rats in each. The Parkinson's disease model was established by micro-injection of 6-hydroxyl-dopamine (6-OHDA) into the right striatum. EA (2 Hz, 1 mA) was applied to "Fengfu"(GV16) and "Taichong" (LR3) for 30 min, once daily for 2 weeks. HE staining, Nissl staining and immunohistochemical staining were conducted for observing the morphological changes of substantia nigra pars compacta (SNc) and striatum, and the tyrosine hydroxylase(TH) immuno-reaction (IR) positive neurons and nerve fibers. The apoptosis was detected by flow cytometry. RESULTS: Compared to the normal control and sham groups, the total number of TH-IR positive neurons and Nissl-stained cells, and OD value of TH-IR positive nerve fibers in the SNc on the 6-OHDA-lesioned side were significantly lower in the model group (P < 0.01). Compared with the model group, the aforementioned 3 indexes of the EA group were increased obviously (P < 0.01). The percentage of apoptosis of SNc on the 6-OHDA-lesioned side was obviously lower in the EA group than in the model group (P < 0.01), but still obviously higher than in the normal group and sham group (P < 0.01). CONCLUSION: Electroacupuncture therapy can significantly increase the number of neurons of substantia nigra and the density of striatum nerve fibers,and reduce the apoptotic percentage of substantia nigra cells in the Parkinsonian rats, which may contribute to its effect in relieving Parkinson's disease.

Optimal frequency of treatment with the 308-nm excimer laser for vitiligo on the face and neck.

OBJECTIVES: This study was designed to determine the optimal treatment frequency with the 308-nm excimer laser for vitiligo and identify key clinical variable(s) associated with treatment efficacy at the optimal frequency. BACKGROUND DATA: Optimal clinical parameters for excimer laser treatment of vitiligo have not been fully determined. Data about the influence on treatment frequency of different clinical variables of vitiligo are needed to facilitate effective treatment regimens. METHODS: A total of 187 patients were treated with the 308-nm excimer laser for 20 sessions at different frequencies (0.5, 1.0, 2.0, and 3.0 per week). The repigmentation rate was graded on a six-point scale and was blindly evaluated by independent physicians. RESULTS: The final percentage of repigmentation for group 0.5 was statistically lower than those for group 1.0, 2.0, and 3.0, and percentages of final levels of repigmentation among these three groups were not statistically different. The clinical variables showed no statistical differences in the final repigmentation effect. Repigmentation occurred fastest with treatment frequencies of 2.0 and 3.0 and there was no statistically significant difference between them. The onset of repigmentation correlated with the area of vitiliginous patches treated, not with the other clinical variables. CONCLUSIONS: The 308-nm excimer laser is effective for therapy to treat vitiligo on the face and neck. The ultimate laser-induced repigmentation effect does not correlate with treatment frequency and repigmentation occurs faster with treatment frequencies of 2.0 and 3.0 than that of 1.0. It appears that the onset of repigmentation correlates with the total area of vitiliginous patches and the optimal treatment frequency. Monitored studies on a larger population with long-term follow-up would be needed to confirm and extend our findings.

[Effects of electroacupuncture on behaviors and dopaminergic neurons in the rat of Parkinson's disease].

OBJECTIVE: To explore the possibility of electroacupuncture (EA) for prevention of the injury of dopaminergic neurons in the substantia nigra of the rat with Parkinson's disease. METHODS: Wistar rats were randomly divided into a normal group, a model group, a sham-operation group and an EA group. 6-OH-DA was injected into right substantia nigra of the midbrain to made Parkinson's disease rat model with single side substantia nigra injury, and TH/TUNEL method and rotation behavior observation method were used to observe changes of rotation behavior and apoptosis of dopaminergic neurons in the substantia nigra after EA at "Taichong" (LR 3) and "Fengfu" (GV 16) for 3 d, 7 d and 14 d. RESULTS: The rotation times/min were same at 3 d, 7 d and 14 d were the basically same in the model group, and at 14 d significantly decreased in the EA group (P<0.05); the rotation starting time at 7 d and 14 d in the model group were significantly longer than those in the EA group (P<0.05); the rotation lasting time at 3 d, 7 d and 14 d in the model group and the EA group were gradually shortened (P<0.01, or P<0.05), but at 7 d, 14 d in the model group were significantly longer than those in the EA group (P<0.05); the DA neuron apoptosis number in the model group were significantly higher than those in the normal group, with a very significant difference (P<0.01); the apoptosis number in the EA group tended to decrease, at 7 d and 14 d were significantly lower than that in the model group (P<0.05). CONCLUSION: Electroacupuncture can effectively prevent from injury of dopaminergic neurons in the substantia nigra of the rat with Parkinson's disease.