[Effect of electroacupuncture pretreatment on ferroptosis in neurons of rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture（EA）preconditioning on ferroptosis in rats with cerebral ischemia-reperfusion injury （CIRI）, so as to explore the neuroprotective mechanism of EA preconditioning. METHODS: Male SD rats were randomly divided into sham operation, model, EA, inhibitor and inducer groups with 20 rats in each group. The CIRI model was established by modified Zea Longa occlusion of the middle cerebral artery. Before modeling, EA treatment （2 Hz/15 Hz, 1-2 mA） was applied to "Baihui"（GV20）, "Fengfu"（GV16） and "Dazhui"（GV14） for rats of the EA group, 20 min a day for 7 consecutive days. Rats of the inhibitor group were intraperitoneally injected with ferristatin-1（25 mg/kg）at a slow and uniform rate. Rats of the inducer group were intraperitoneally injected with Erastin（100 mg/kg） after 7 days of EA preconditioning, once every 2 h for a total of 4 times. The CIRI models were prepared 2 d later after the above interventions finished by thread-occlusion. The degree of neurological impairment was evaluated by modified Zea Longa score. The percentage of infarct size was calculated by TTC staining. The ultrastructure of neurons in hippocampus was observed by transmission electron microscope. The contents of ferrous ion （Fe2+）, malondialdehyde （MDA） and glutathione （GSH） in cerebral tissue and reactive oxygen species （ROS） in serum were determined by biochemical method. The changes of mitochondrial membrane potential in rats brain tissues were detected by flow cytometry. The mRNA and protein expression levels of glutathione peroxidase 4 （GPX4）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, transferrin receptor （TFRC）, 15-lipoxygenase （15-LOX） and cyclooxygenase-2 （COX-2） in the ischemic hippocampal region of CIRI rats were detected by real-time quantitative PCR and Western blot, respectively. RESULTS: Compared with the sham operation group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression levels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01）, and mitochondria in brain tissue were significantly damaged （P<0.01） in the model group. Compared with the model group, the above indexes were all reversed （P<0.05, P<0.01） in the EA group and inhibitor group. Compared with the EA group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression le-vels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.05, P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01, P<0.05）, and mitochondria in brain tissue were significantly damaged （P<0.05） in the inducer group. CONCLUSION: EA preconditioning has neuroprotective effect on CIRI rats, which may be related to inhibiting ACSL4/TFRC/15-LOX/COX-2 expression and increasing GSH/GPX4 expression.

[Effect of Tongdu Tiaoshen electroacupuncture pretreatment on PPARγ-mediated pyroptosis of cerebral cortex in rats with cerebral ischemia reperfusion injury].

OBJECTIVE: To observe the effect of Tongdu Tiaoshen (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI. METHODS: A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1ß and IL-18 in the cerebral cortex of rats were determined by ELISA. RESULTS: Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (P<0.01); and the protein expression of GSDMD-N and contents of IL-1ß and IL-18 were increased (P<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1ß and IL-18 were lower (P<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (P<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (P<0.01, P<0.05), the mRNA expression of caspase-1 was reduced (P<0.01); and the contents of IL-1ß and IL-18 were lower (P<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.05, P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1ß and IL-18 were declined (P<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.05, P<0.01), the mRNA expression of PPARγ was decreased (P<0.01) and the protein expression of GSDMD-N was elevated (P<0.05); and the contents of IL-1ß and IL-18 were higher (P<0.01). CONCLUSION: Tongdu Tiaoshen EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.

[Professor HAN Wei's clinical experience of acupuncture and moxibustion with Tongyang Xingshen for adolescent depressive disorder].

Professor HAN Wei 's clinical experience of acupuncture and moxibustion with Tongyang Xingshen (promoting yang and regaining consciousness) for adolescent depressive disorder is introduced. It is believed that the internal causes of adolescent depressive disorder are mostly emotional and physical factors, while the external causes are mainly social factors, and yang-qi stagnation and emotional disorder are the key pathogenesis. The key of acupuncture and moxibustion with Tongyang Xingshen is warming and regulating the governor vessel. The governor vessel acupoints at head, neck and back are selected. At head, Baihui (GV 20) and Yintang (GV 24+) are selected; at neck, Fengfu (GV 16) and Dazhui (GV 14) are selected; at back, Taodao (GV 13), Shenzhu (GV 12), Shendao (GV 11), Zhiyang (GV 9) and Jinsuo (GV 8) are selected. The combination of disease differentiation and syndrome differentiation should be highly valued, and the moxibustion with Tongyang and acupuncture with Xingshen should be used simultaneously, and the strong stimulation is suggested.

[Acupuncture combined with Qingfei Qutan decoction for stroke-associated pneumonia with phlegm-heat obstructing lung and its effect on cellular immune function].

OBJECTIVE: To observe the clinical effect of acupuncture combined with Qingfei Qutan decoction for stroke-associated pneumonia (SAP) with phlegm-heat obstructing lung, and explore its possible mechanism. METHODS: Ninety-nine patients of SAP with phlegm-heat obstructing lung were randomly divided into a combination group (33 cases, 1 case dropped off), a Chinese medication group (33 cases, 1 case dropped off) and an acupuncture group (33 cases, 1 case dropped off). On the basis of routine basic treatment, the patients in the acupuncture group were treated with acupuncture at Tiantu (CV 22), Feishu (BL 13), Taiyuan (LU 9), Sanyinjiao (SP 6), etc., once a day, with an interval of 1 day after continuous 6-day treatment; the patients in the Chinese medication group were treated with Qingfei Qutan decoction, 1 dose per day; the patients in the combination group were treated with acupuncture combined with Qingfei Qutan decoction. Two weeks were taken as a course of treatment, and two courses of treatment were given. Before and after treatment, the clinical pulmonary infection score (CPIS), inflammatory indexes (neutrophil-to-lymphocyte ratio [NLR], procalcitonin [PCT], C-reactive protein [CRP]), cellular immune function (CD+3, CD+4, CD+8 and CD+4/CD+8) were compared in the 3 groups. The clearance of pathogenic bacteria after treatment was observed in the 3 groups. The clinical efficacy of each group was evaluated. RESULTS: After treatment, the CPIS scores, NLR, PCT, CRP and CD+8 in the each group were lower than those before treatment (P<0.05), while the levels of CD+3, CD+4, CD+4/CD+8 were higher than those before treatment (P<0.05). The above indexes in the combination group were better than those in the acupuncture group and the Chinese medication group (P<0.05), and the above indexes in the Chinese medication group were better than those in the acupuncture group (P<0.05). There was no significant difference in the clearance rate of pathogenic bacteria among three groups (P>0.05). The cured and markedly effective rate was 65.6% (21/32) in the combination group, which was higher than 43.8% (14/32) in the Chinese medication group and 18.8% (6/32) in the acupuncture group (P<0.05). The cured and markedly effective rate in the Chinese medication group was higher than that in the acupuncture group (P<0.05). CONCLUSION: Acupuncture combined with Qingfei Qutan decoction could effectively improve the clinical symptoms of SAP patients with phlegm-heat obstructing lung, and the mechanism may be related to enhancing the cellular immune function and reducing the level of inflammatory reaction.

[Tongdu Tiaoshen acupuncture combined with carotid endarterectomy for carotid artery stenosis: a randomized controlled trial].

OBJECTIVE: To observe the clinical therapeutic effect of Tongdu Tiaoshen acupuncture combined with carotid endarterectomy (CEA) and simple CEA on carotid artery stenosis (CAS). METHODS: A total of 60 patients with CAS were randomized into an observation group (30 cases, 2 cases dropped off) and a control group (30 cases, 3 cases dropped off). Both groups were treated with eversion CEA (eCEA). The conventional treatment of internal medicine and antiplatelet drugs i.e. aspirin enteric-coated tablet and clopidogrel hydrogen sulfate tablet were given in the control group for 4 weeks. On the basis of the treatment in the control group, Tongdu Tiaoshen acupuncture was applied at Baihui (GV 20), Fengfu (GV 16), Yamen (GV 15), cervical Jiaji (EX-B 2), Dazhui (GV 14), etc. in the observation group, once a day, 1-day rest was taken after 6-day treatment, 2 weeks were as one course and totally 2 courses were required. The carotid intima-media thickness (IMT) before and after treatment was detected by ultrasonic diagnostic apparatus, the TCM symptom score was compared before and after treatment and in the follow-up of 6 months after treatment, the clinical efficacy was evaluated in the two groups. The occurrence of endpoints within 1 year was recorded. RESULTS: After treatment, the carotid IMT and TCM symptom scores were decreased compared before treatment in the both groups (P<0.05), and the changes in the observation group were greater than the control group (P<0.05). In the follow-up, the TCM symptom scores were decreased compared before treatment in the both groups (P<0.05). The total effective rate was 96.4% (27/28) in the observation group, which was superior to 88.9% (24/27) in the control group (P<0.05). There were 1 case of stoke in the observation group and 2 cases of stroke in the control group within 1-year follow-up, and there was no significant difference in the number of endpoints between the two groups within 1 year (P>0.05). CONCLUSION: Tongdu Tiaoshen acupuncture combined with CEA can effectively reduce the IMT in patients with CAS, improve the TCM symptom score, the efficacy is superior to simple CEA treatment.

[Exploration on the diagnosis and treatment of generalized myasthenia gravis with acupuncture and moxibustion based on the study of ancient medical works].

Through collecting the relevant provisions and medical cases of wei syndrome treated with acupuncture and moxibustion from ancient medical works, the diagnosis and acupoint selection in treatment of generalized myasthenia gravis (gMC) with acupuncture and moxibustion were analyzed systematically from 3 aspects, i.e. meridian differentiation, disease differentiation and syndrome differentiation. In treatment based on meridian differentiation, the acupoints are selected in the light of the running course of meridian and characteristics of meridian disorders. In treatment based on disease differentiation, the acupoints are selected in accordance with etiology, pathogenesis and transmission stages of wei syndrome. Concerning to syndrome differentiation in treatment, the acupoints are selected on the basis of therapeutic principles determined by different syndromes/patterns of wei syndrome. In modern clinical practice, the treatment for gMC should be rooted at ancient literature, thus a standardized regimen can be developed for diagnosis and treatment with acupuncture and moxibustion.

[Effect of moxibustion on expression of GAP-43 in sciatic nerve trunk and ventral horn of spinal cord of rats with primary sciatica and its nerve repair function].

OBJECTIVE: To observe the effect of moxibustion at "Huantiao" (GB 30) on the expression of growth-associated protein-43 (GAP-43) in the sciatic nerve trunk and ventral horn of spinal cord (L4-L6) in rats with primary sciatica, and to explore the mechanism of moxibustion in improving primary sciatica. METHODS: A total of 48 healthy male SD rats were randomly divided into a normal group, a sham operation group, a model group and a moxibustion group, 12 rats in each group. The rat model of primary sciatic pain was established by chronic constriction injury (CCI) of the sciatic nerve in the model group and the moxibustion group. On the 8th day of the experiment, moxibustion was adopted at "Huantiao" (GB 30) in the moxibustion group for 5-10 min, once a day for 14 consecutive days. Sciatic nerve function index (SFI) was measured and compared in each group at day 1, 7, 14 and 21. On the 21st day of the experiment, HE staining was used to observe the morphology of ventral horn of rat spinal cord and sciatic nerve trunk. Immunohistochemical method and real-time PCR were used to detect mRNA and protein expressions of GAP-43 in the spinal cord and sciatic nerve trunk of rats. RESULTS: On day 7, 14 and 21, there was no statistical difference in SFI between the sham operation group and the normal group (P>0.05); compared with the sham operation group on day 7, 14 and 21, the SFI of the model group was reduced (P<0.01); compared with the model group on day 14 and 21, SFI in the moxibustion group was increased (P<0.01). In the normal group and the sham operation group, neuronal cells were in order in the ventral horn of the spinal cord, nissl bodies were spaced regularly, the myelin sheath structure of sciatic nerve axon was clearly visible. In the model group, neuronal cells were deformed and ruptured in the ventral horn of the spinal cord, the number of nissl bodies was less, and the demyelination of sciatic axons appeared. In the moxibustion group, neuronal cells were found in the ventral horn of spinal cord, and the number of nissl bodies was increased, and less demyelinating changes of axons appeared in sciatic nerve. Compared with the normal group, the expressions of GAP-43 mRNA and GAP-43 protein in the sciatic nerve trunk and GAP-43 protein in the ventral horn of spinal cord were increased in the sham operation group (P<0.01). Compared with the sham operation group, the expression of GAP-43 mRNA and GAP-43 protein in the spinal cord and sciatic nerve trunk of rats in the model group was increased. Compared with the model group, the expression of GAP-43 mRNA and GAP-43 protein in the spinal cord and sciatic nerve trunk of rats in the moxibustion group was increased (P<0.01). CONCLUSION: Moxibustion at "Huantiao" (GB 30) could improve the sciatic nerve function in rats with primary sciatica and its mechanism may be related to improving the expression of GAP-43 and enhancing the self-repair ability of the sciatic nerve after injury.

[Moxibustion at acpoints of governor vessel on regulating PI3K/Akt/mTOR signaling pathway and enhancing autophagy process in APP/PS1 double-transgenic Alzheimer's disease mice].

OBJECTIVE: To observe the eliminating effects of moxibustion at "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) on amyloid ß-peptide (Aß) in brain of the amyloid precursor protein/presenili1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD) by regulating the phosphoinositide 3-kinases/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. METHODS: A total of 60 APP/PS1 double-transgenic mice with AD were randomly divided into a model group, a moxibustion group, a rapamycin group and a combination group (treated with moxibustion and inhibitor), 15 mice in each group, another 15 male C57BL/6J mice with same age and background were selected as the control group. In the moxibustion group, pressing moxibustion was applied at "Baihui" (GV 20) while the mild moxibustion was applied at "Fengfu" (GV 16) and "Dazhui" (GV 14). The treatment was manipulated for 20 min each time, once a day for 2 weeks. In the rapamycin group, rapamycin (2 mg/kg) was given by intraperitoneal injection once a day for 2 weeks. On the basis of the treatment in the moxibustion group, 3-methyladenine (1.5 mg/kg) was given by intraperitoneal injection once a day for 2 weeks. The mice in the control and the model group received normal diet and no intervention was given for 2 weeks. Immunohistochemica method was used to measure the levels of Aß1-42 in the cerebral cortex and hippocampal, transmission electron microscopy was used to observe the formation of autophagosome in hippocampus, and Western blot method was used to observe the levels of PI3K, Akt, p-Akt, mTOR and p-mTOR in hippocampus. RESULTS: Compared with the control group, the levels of Aß1-42 in the cerebral cortex and hippocampal were increased in the model group (P<0.01). Compared with the model group, the levels of Aß1-42 in the cerebral cortex and hippocampal were decreased in the moxibustion group, the rapamycin group and the combination group (all P<0.01), compared with the moxibustion group, the levels of Aß1-42 in the cerebral cortex and hippocampal were increased in the combination group (P<0.01), while there was no significant difference between the moxibustion group and the rapamycin group in the levels of Aß1-42（P>0.05）. Compared with the rapamycin group, the levels of Aß1-42 in the cerebral cortex and hippocampal were increased in the combination group (P<0.01). In the model group, the cytoplasmic utophagic vacuoles and organelles of neuron were reduced. In the moxibustion group, the utophagic vacuoles were increased, and the organelles showed deformation and atrophy. In the rapamycin group, the utophagic vacuoles were widely disturbed and few deformed organelles were found. In the combination group, few utophagic vacuoles were found and additional organelles showed deformation and atrophy. Compared with the control group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the model group (all P<0.01). Compared with the model group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were reduced in the moxibustion group, the rapamycin group and the combination group (all P<0.01). Compared with the moxibustion group, the levels of PI3K、Akt、and p-mTOR were increased in the rapamycin group and the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the combination group (all P<0.01). Compared with the rapamycin group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the combination group (P<0.01). CONCLUSION: Moxibustion at acupoints of governor vessel can enhance the autophagy process on Aß1-42 in brain of the APP/PS1 double-transgenic AD mice, which may be associated with its effects on inhibiting the abnormal activation of PI3K/Akt/mTOR signaling pathway.

Role of the PERK-eIF2α-CHOP Signaling Pathway in the Effect of Needle Knife Therapy on Knee Joint Chondrocyte Apoptosis.

Needle knife therapy, a form of acupuncture and moxibustion, has been widely used in the clinical treatment of knee osteoarthritis (KOA). However, the mechanism is not clear. Therefore, we studied the mechanisms of action of needle knife intervention on KOA in rabbits, with the PERK-eIF2α-CHOP pathway as a starting point, in order to determine the mechanism underlying knee joint chondrocyte apoptosis. Apoptosis and ultrastructural changes in the articular cartilage were examined by pathological study and transmission electron microscopy, and PERK, eIF2α, and CHOP mRNA and protein levels were detected by qRT-PCR and western blot, respectively. PERK, eIF2α, and CHOP protein levels were significantly higher in the model group than in the normal group (P < 0.01) and were considerably downregulated in the needle knife and the medicine groups compared to the model group (P < 0.01). The eIF2α, p-eIF2α, and CHOP protein levels were not significantly different between the needle knife and medicine groups. The PERK, eIF2α, and CHOP mRNA levels in the drug group were higher than those in the needle knife group (P < 0.01). Needle knife therapy can regulate PERK-eIF2α-CHOP signaling pathway, which could be one of the mechanisms by which it affects chondrocyte apoptosis in KOA rabbits.

[Moxibustion improves learning-memory ability by promoting cellular autophagy and regulating autophagy-related proteins in hippocampus and cerebral cortex in APP/PS1 transgenic Alzheimer's disease mice].

OBJECTIVE: To observe the effect of moxibustion of acupoints of the Governor Vessel on the levels of cellular autophagy, ß amyloid protein (Aß) immunoactivity, and expression of LC3-Ⅰ， LC3-Ⅱ， p62 and p-P70S6K proteins in the hippocampal tissue of APPswe/PS1de9 (APP/PS1) double-transgenic Alzheimer's disease (AD) mice, so as to reveal its underlying mechanisms in improving AD. METHODS: APP/PS1 double-transgenic AD mice were randomly divided into AD model, moxibustion, autophagy-inducer (Rapamycin) and autophagy-inhibitor (3-MA)＋moxibustion groups (n＝10 in each group), and other 10 C57BL/6J male mice (the same age) were used as the normal control group. Herbal-cake (made of Chuanwu [Radix Aconiti Praeparata]) partitioned moxibustion was applied to "Baihui"(GV20), moxibustion was applied to "Fengfu"(GV16) and "Dazhui"(GV14), all for 20 min, once daily for 2 weeks, with one day's off between two weeks. For mice of the autophagy-inducer and 3-MA＋moxibustion groups, Rapamycin (2 mg•kg－1•d－1) and 3-MA (1.5 mg•kg－1•d－1) were separately administered by intraperitoneal injection for 2 weeks. The cognitive ability was examined by Morris water maze tests, and the ultrastructural changes (including autophagic lysosomes, etc.) of hippocampal neurons were observed by using transmission electron microscopy. The immunoactivity of cerebral cortex and hippocampal Amyloid ß peptide 1-42 (Aß1-42) was detected by immunohistochemistry, and the expression levels of hippocampal LC3-Ⅰ， LC3-Ⅱ， p62 and p-P70S6K proteins were detected by Western blot. RESULTS: After modeling, the escape latency of Morris water maze tasks was prolonged in the model group than in the normal control group (P<0.05) and obviously shortened in the moxibustion and autophagy-inducer groups (not the autophagy-inhibitor group) than in the model group (P<0.05). Results of transmission electron microscope showed deformed, irregular or atrophic neurons with rough and incomplete and fuzzy nuclear membrane, and decreased intracellular autophagosomes in the hippocampus in the model group, and partial irregular, atrophic neurons with more autophagic vesicles and lysosomes in the moxibustion group. The expression levels of Aß1-42 in both cerebral cortex and hippocampus tissues, and LC3-Ⅰ， p62 and p-P70S6K proteins in the hippocampus were consi-derably up-regulated in the model group relevant to the normal control group (P<0.01), and evidently down-regulated in both moxibustion and autophagy-inducer groups (not the autophagy-inhibitor group) than in the model group (P<0.01), while that of hippocampal LC3-Ⅱ protein and LC3-Ⅱ/Ⅰ ratio levels were obviously down-regulated in the model group relevant to the normal control group (P<0.01), and significantly up-regulated in both moxibustion and autophagy-inducer groups (not the autophagy-inhibitor group) than in the model group (P<0.01)．. CONCLUSION: Moxibustion can improve the cognitive ability of APP/PS1 double-transgenic AD mice, which is associated with its effects in promoting hip-pocampal and cerebral cortex autophagy level, and down-regulating the expression levels of Aß1-42, LC3-Ⅰ， p62 and p-P70S6K proteins in the hippocampus.

Regulation of the endoplasmic reticulum stress response and neuroprotective effects of acupuncture on brain injury caused by heroin addiction.

OBJECTIVES: To evaluate regulation of the endoplasmic reticulum stress (ERS) response by acupuncture and to investigate its neuroprotective effect on brain injury caused by heroin addiction. METHODS: A total of 48 male Sprague-Dawley rats were randomly divided into a healthy control group (Control), an untreated heroin exposed group (Heroin) and a heroin exposed group receiving electroacupuncture (EA) treatment at GV14 and GV20 (Heroin+acupuncture) with n=16 rats per group. A rat model of heroin addiction was established by intramuscular injection of incremental doses of heroin for 8 consecutive days. A rat model of heroin relapse was established according to the exposure (addiction) → detoxification method. Apoptotic changes in nerve cells in the hippocampus and ventral tegmental area (VTA) were evaluated in each group of rats using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. PERK, eIF2a, CHOP, IRE1 and JNK gene expression and protein expression were measured using quantitative real-time PCR (RT-qPCR) assay and immunohistochemical assay, respectively. RESULTS: The total number of positive nerve cells in the hippocampus and VTA was significantly lower in the Heroin+acupuncture group than in the Heroin group (p<0.01). Compared with the Heroin group, mRNA and protein expression of PERK, eIF2a, CHOP, IRE1 and JNK in the hippocampus and VTA were significantly downregulated in the Heroin+acupuncture group (p<0.05). CONCLUSION: The acupuncture-regulated ERS response appears to mediate the neuroprotective effect of acupuncture in heroin-addicted rats with brain injury. Inhibition of CHOP and JNK upregulation and reduction of nerve cell apoptosis may be the main mechanisms underlying the effects of acupuncture on heroin addiction-induced brain injury.

Hyperbaric Oxygen and Ginkgo Biloba Extract Ameliorate Cognitive and Memory Impairment via Nuclear Factor Kappa-B Pathway in Rat Model of Alzheimer's Disease.

BACKGROUND: Hyperbaric oxygen (HBO) and Ginkgo biloba extract (e.g., EGB 761) were shown to ameliorate cognitive and memory impairment in Alzheimer's disease (AD). However, the exact mechanism remains elusive. The aim of the present study was to investigate the possible mechanisms of HBO and EGB 761 via the function of nuclear factor kappa-B (NF-κB) pathway. METHODS: AD rats were induced by injecting ß-amyloid 25-35 into the hippocampus. All animals were divided into six groups: Normal, sham, AD model, HBO (2 atmosphere absolute; 60 min/d), EGB 761 (20 mg·kg-1·d-1 ), and HBO/EGB 761 groups. Morris water maze tests were used to assess cognitive, and memory capacities of rats; TdT-mediated dUTP Nick-End Labeling staining and Western blotting were used to analyze apoptosis and NF-κB pathway-related proteins in hippocampus tissues. RESULTS: Morris water maze tests revealed that EGB 761 and HBO significantly improved the cognitive and memory ability of AD rats. In addition, the protective effect of combinational therapy (HBO/EGB 761) was superior to either HBO or EGB 761 alone. In line, reduced apoptosis with NF-κB pathway activation was observed in hippocampus neurons treated by HBO and EGB 761. CONCLUSIONS: Our results suggested that HBO and EGB 761 improve cognitive and memory capacity in a rat model of AD. The protective effects are associated with the reduced apoptosis with NF-κB pathway activation in hippocampus neurons.

[Cloning full-length homologous cDNAs of pollen allergens in Humulus Scandens(Lour.) Merr by degenerate primer].

AIM: To establish a stable and reliable method for fast cloning homologous genes of pollen allergens in allergen-containing plants. METHODS: Degenerate primers were designed based on the bioinformatic analysis of numerous allergens available from the database. Subsequent amplification of the allergen genes was conducted in the weed pollen cDNA pool by a selective PCR profile. Following the truncated gene cloning, RACE method was used to isolate full-length cDNA. Gene function was deduced by sequence alignment in GenBank database. The degenerate ability of the primer was compared with the full-length cDNA sequences. RESULTS: Three full-length cDNAs were obtained. Sequence analysis showed that these new genes shared as high as 79%-85% homology with a large amount of known allergen profilins and were hence regarded as members of panallergen profilin family. Comparing these genes with the degenerate primers that were initially used in truncated gene cloning revealed that alternative nucleotide degeneracy occurred beyond the degenerate site predesigned, suggesting that further degeneracy was expanded by Touchdown-gradient PCR. CONCLUSION: Cloning of homologous genes or allergen genes can be efficiently achieved by using the combination of degenerate primer with Touchdown-gradient RT-PCR in the species such as Humulus scandens that has not yet been investigated.