Electroacupuncture protects the intestinal mucosal barrier in diarrhea-predominant Irritable Bowel Syndrome rats by regulating the MCs/Tryptase/PAR-2/MLCK pathway.

OBJECTIVE: The pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D) is related to damage to the intestinal mucosal barrier function. Based on the Mast cell (MC)/Tryptase/Protease-activated receptor-2 (PAR-2)/Myosin light chain kinase (MLCK) pathway, this study explored the effect of electroacupuncture (EA) on IBS-D rats and its possible mechanism of protecting the intestinal mucosal barrier. METHODS: The IBS-D rat model was established by mother-offspring separation, acetic acid enema, and chronic restraint stress. The efficacy of EA on IBS-D rats was evaluated by observing the rate of loose stool (LSP) and the minimum volume threshold of abdominal withdrawal reflex (AWR) in rats. Mast cells and the ultrastructure of intestinal mucosa were observed by H&E staining, toluidine blue staining, and transmission electron microscopy. The expression levels of Tryptase, PAR-2, MLCK, zonula occludens-1 (ZO-1), and Occludin in rats were detected by ELISA, qRT-PCR, and western blot. RESULTS: After 7 days of intervention, compared to the IBS-D group, the loose stool rates of rats in IBS-D + EA group and IBS-D + ketotifen group were decreased (P < 0.01), the minimum volume thresholds of AWR were improved (P < 0.01), the inflammation of colon tissue decreased, the number of MCs were decreased (P < 0.01), the expression of Tryptase, PAR-2, and MLCK were lowered (P < 0.01, P < 0.05), and the expression of ZO-1 and Occludin were enhanced (P < 0.01, P < 0.05). Compared to the EA group, there was no significant difference in each index between the ketotifen groups (P > 0.05). CONCLUSION: EA has a good therapeutic effect on IBS-D rats. Regulating the MCs/Tryptase/PAR-2/MLCK pathway may be a mechanism to protect the intestinal mucosal barrier.

Effect of moxibustion on colonic low-grade inflammatory response in rats with diarrhea-predominant irritable bowel syndrome based on mast cell degranulation. / åŸºäºŽè‚¥å¤§ç»†èƒžè„±é¢—ç²’æŽ¢è®¨è‰¾ç¸å¯¹è ¹æ³»åž‹è‚ æ˜“æ¿€ç»¼åˆå¾å¤§é¼ ç»“è‚ ä½Žåº¦ç‚Žæ€§ååº”çš„å½±å“.

OBJECTIVES: To observe the effects of moxibustion on colonic mast cell degranulation and inflammatory factor expression in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), and explore the potential mechanism of moxibustion in treating IBS-D. METHODS: Forty-five rat pups born from 5 healthy SPF-grade pregnant SD rats, with 8 rats were randomly selected as the normal group. The remaining 37 rats were intervened with maternal separation, acetic acid enema, and chronic restraint stress to establish the IBS-D model. The successfully modeled 32 rats were then randomly assigned to a model group, a ketotifen group, a moxibustion group, and a moxibustion-medication group, with 8 rats in each group. The rats in the ketotifen group were intervened with intragastric administration of ketotifen solution (10 mL/kg); the rats in the moxibustion group were intervened with suspended moxibustion on bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37); the rats in the moxibustion-medication group were intervened with suspended moxibustion combined with intragastric administration of ketotifen solution. All interventions were administered once daily for 7 consecutive days. The diarrhea rate and minimum volume threshold of abdominal withdrawal reflex (AWR) were calculated before and after modeling, as well as after intervention. After intervention, colonic tissue morphology was observed using HE staining; colonic mucosal ultrastructure was examined by scanning electron microscopy; colonic mast cell ultrastructure was observed using transmission electron microscopy; mast cell degranulation was assessed by toluidine blue staining; serum and colonic levels of histamine, interleukin (IL)-1ß, IL-6, IL-1α, trypsin-like enzyme, and protease-activated receptor 2 (PAR-2) were measured by ELISA; the Western blot and real-time quantitative PCR were employed to evaluate the protein and mRNA expression of colonic IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2; the immunofluorescence was used to detect the positive expression of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colonic tissue. RESULTS: Compared to the normal group, the rats in the model group exhibited extensive infiltration of inflammatory cells in colonic tissue, severe damage to the colonic mucosa, disordered arrangement of villi, reduced electron density, and a significant decrease in granule quantity within mast cells. The diarrhea rate and mast cell degranulation rate were increased (P<0.01), AWR minimum volume threshold was decreased (P<0.01); the serum and colonic levels of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were elevated (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all elevated (P<0.01). Compared to the model group, the rats in the ketotifen group, the moxibustion group, and the moxibustion-medication group exhibited significantly reduced infiltration of inflammatory cells in colonic tissue, relatively intact colonic mucosa, orderly arranged villi, increased electron density, and an augmented number of mast cell granules; the diarrhea rate and mast cell degranulation rate were decreased (P<0.01), and AWR minimum volume threshold was increased (P<0.01); the serum and colonic levels of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were reduced (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all decreased (P<0.01). Compared to the ketotifen group, the moxibustion group showed decreased serum levels of histamine, IL-6, and trypsin-like enzyme (P<0.01, P<0.05), as well as reduced colonic levels of IL-1ß and IL-6 (P<0.01, P<0.05); the protein expression of colonic IL-1ß, IL-1α, and PAR-2 was reduced (P<0.05), and the positive expression of colonic IL-1ß and trypsin-like enzyme was reduced (P<0.01, P<0.05). Compared to both the ketotifen group and the moxibustion group, the moxibustion-medication group exhibited decreased diarrhea rate and mast cell degranulation rate (P<0.01), an increased AWR minimum volume threshold (P<0.01), reduced serum and colonic levels of histamine, IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01), decreased protein expression of colonic IL-1ß, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), reduced mRNA and positive expression of colonic IL-1ß, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), and decreased positive expression of colonic histamine (P<0.01). CONCLUSIONS: Moxibustion on "Tianshu" (ST 25) and "Shangjuxu" (ST 37) might inhibit low-grade inflammatory reactions in the colon of IBS-D model rats. The mechanism may be related to the inhibition of histamine and trypsin-like enzyme secreted by mast cell, thereby reducing the expression of related inflammatory factors.

Enhancing operational stability of OLEDs based on subatomic modified thermally activated delayed fluorescence compounds.

The realization of operationally stable blue organic light-emitting diodes is a challenging issue across the field. While device optimization has been a focus to effectively prolong device lifetime, strategies based on molecular engineering of chemical structures, particularly at the subatomic level, remains little. Herein, we explore the effect of targeted deuteration on donor and/or acceptor units of thermally activated delayed fluorescence emitters and investigate the structure-property relationship between intrinsic molecular stability, based on isotopic effect, and device operational stability. We show that the deuteration of the acceptor unit is critical to enhance the photostability of thermally activated delayed fluorescence compounds and hence device lifetime in addition to that of the donor units, which is commonly neglected due to the limited availability and synthetic complexity of deuterated acceptors. Based on these isotopic analogues, we observe a gradual increase in the device operational stability and achieve the long-lifetime time to 90% of the initial luminance of 23.4 h at the luminance of 1000 cd m-2 for thermally activated delayed fluorescence-sensitized organic light-emitting diodes. We anticipate our strategic deuteration approach provides insights and demonstrates the importance on structural modification materials at a subatomic level towards prolonging the device operational stability.

[Effect of needle-knife on chondrocyte apoptosis of knee joint in rabbits with knee osteoarthritis based on CircSERPINE2-miR-1271-5P-ERG axis].

OBJECTIVE: To observe the effect of needle-knife on the chondrocyte apoptosis of knee joint in rabbits with knee osteoarthritis (KOA) based on the CircSERPINE2-miR-1271-5P-E26 specific transformation-related gene (ERG) axis, and to explore the mechanism of needle-knife for KOA. METHODS: Thirty-six New Zealand white rabbits were randomly divided into a normal group, a model group, a needle-knife group and a sham needle-knife group, 9 rabbits in each group. The rabbits in the model group, the needle-knife group and the sham needle-knife group were treated with modified Videman method to prepare KOA model. After successful modeling, the rabbits in the needle-knife group were treated with needle-knife at cord adhesion and nodules near quadriceps femoris tendon and internal and external collateral ligament on the affected knee joint; the rabbits in the sham needle-knife group were treated with sham needle-knife baside the needle insertion point of the needle-knife group (needle-knife was only inserted, without any operation). The treatment was given once a week, 3 times in total. The Lequesne MG behavioral score was used to evaluate the knee joint damage in each group before and after intervention. After intervention, HE staining and transmission electron microscopy were used to observe the cartilage tissue morphology and ultrastructure of chondrocytes in the knee joint in each group; TUNEL method was used to detect the level of chondrocyte apoptosis in the knee joint; real-time fluorescence quantitative PCR was used to detect the expression of CircSERPINE2, miR-1271-5P and ERG mRNA in knee cartilage tissue in each group. RESULTS: After intervention, compared with the normal group, the Lequesne MG behavioral score in the model group was increased (P<0.01). Compared with the model group and the sham needle-knife group, the Lequesne MG behavioral score in the needle-knife group was decreased (P<0.01). In the model group and the sham needle-knife group, the number of chondrocytes and organelles was decreased, the cell nucleus was shrunk, mitochondria was swelling or disappeared; in the needle-knife group, the number of chondrocytes and organelles was increased, the cell nucleus was not obviously shrunk and the mitochondria was not obviously swelling. Compared with the normal group, the level of chondrocyte apoptosis in the model group was increased (P<0.01); compared with the model group and the sham needle-knife group, the level of chondrocyte apoptosis in the needle-knife group was decreased (P<0.01, P<0.05). Compared with the normal group, the expression of CircSERPINE2 and ERG mRNA in the model group was decreased (P<0.01), and the expression of miR-1271-5P mRNA was increased (P<0.01); compared with the model group and the sham needle-knife group, the expression of CircSERPINE2 and ERG mRNA in the needle-knife group was increased (P<0.01), and the expression of miR-1271-5P mRNA was decreased (P<0.01). CONCLUSION: Needle-knife could reduce the knee joint damage and chondrocyte apoptosis in KOA rabbits, which may be related to up-regulating the expression of CircSERPINE2 and ERG mRNA, and inhibiting the expression of miR-1271-5P mRNA.

[Effect of moxibustion on autophagy in mice with Alzheimer's disease based on mTOR/p70S6K signaling pathway].

OBJECTIVE: To investigate the effect of moxibustion on autophagy and amyloid ß-peptide1-42 (Aß1-42) protein expression in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD). METHODS: After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aß1-42 protein in frontal cortex and hippocampal tissue was detected by immunohistochemistry; the expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), p70 ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6K (p-p70S6K) protein in hippocampus were detected by Western blot method. RESULTS: Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the model group were increased (P<0.05); compared with the model group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group, rapamycin group and inhibitor group were decreased (P<0.05); compared with the inhibitor group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group and rapamycin group were decreased (P<0.05); compared with the rapamycin group, the protein expressions of mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group were decreased (P<0.05). CONCLUSION: Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aß aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.

[Effect of moxibustion on cognitive function and proteins related with apoptosis of hippocampal neurons in rats with vascular dementia].

OBJECTIVE: To observe the effect of moxibustion on proteins related with apoptosis of hippocampal neurons in rats with vascular dementia (VD), and to explore the possible mechanism of moxibustion on improving VD. METHODS: Thirty SD rats were selected from 100 rats (3 rats were excluded) and randomly divided into a normal group and a sham operation group, 15 rats in each group. The remaining 67 rats were treated with ischemia-reperfusion method at bilateral common carotid artery to establish VD model. The 45 rats with successful VD model were randomly divided into a model group, a moxibustion group and a medication group, 15 rats in each group. On the 7th day after successful modeling, the rats in the moxibustion group were treated with suspended moxibustion at "Guanyuan" (CV 4), "Mingmen" (GV 4) and "Dazhui" (GV 14), 15 min per acupoint, once a day; there was 1 d of rest after 6 d of moxibustion, and the treatment was given for 4 weeks. The rats in the medication group was treated with nimodipine tablets by gavage, 2 mg/kg per day, 3 times a day for 4 weeks. Before and after intervention, the Morris water maze test was used to detect the escape latency of rats in each group; after the intervention, the TUNEL method was used to detect the apoptosis rate of neurons in hippocampal CA1 area; the immunofluorescence double labeling method was used to detect the number of co-expression positive cells of B-cell lymphoma-2 (Bcl-2)/neuronal nuclear antigen (NeuN) and Bcl-2-associated X protein (Bax)/NeuN in hippocampal CA1 area; the immunofluorescence single labeling method was used to detect cytochrome C (cytC) and outer mitochondrial membrane receptor Tom20 (Tom20) in hippocampal CA1 area; the Western blot method was used to detect the p53 upregulated modulator of apoptosis (PUMA) in hippocampus. RESULTS: Before intervention, compared with the normal group and the sham operation group, the escape latency in the model group, the moxibustion group and the medication group was prolonged (P<0.01). After intervention, the escape latency in the moxibustion group and the medication group was shorter than that before intervention (P<0.01). Compared with the model group, the escape latency in the moxibustion group and the medication group was shortened (P<0.05); compared with the medication group, the escape latency in the moxibustion group was shortened (P<0.05). Compared with the normal group and the sham operation group, the apoptosis rate of neurons in hippocampal CA1 area was increased, the number of Bcl-2/NeuN co-expression positive cells was decreased, and the number of Bax/NeuN co-expression positive cells was increased in the model group (P<0.01); compared with the model group, the apoptosis rates of hippocampal CA1 neurons were decreased, the number of Bcl-2/NeuN co-expression positive cells was increased, and the number of Bax/NeuN co-expression positive cells was decreased in the moxibustion group and the medication group (P<0.01); compared with the medication group, the apoptosis rate of neurons in hippocampal CA1 area was decreased, the number of Bcl-2/NeuN co-expression positive cells was increased, and the number of Bax/NeuN co-expression positive cells was decreased in the moxibustion group (P<0.01, P<0.05). Compared with the normal group and the sham operation group, the expressions of cytC, Tom20 protein in hippocampal CA1 area and PUMA protein in hippocampal tissue in the model group were increased (P<0.01); compared with the model group, the expressions of cytC, Tom20 protein in hippocampal CA1 area and PUMA protein in hippocampal tissue in the moxibustion group and the medication group were decreased (P<0.01); compared with the medication group, the expressions of cytC, Tom20 protein in hippocampal CA1 area and PUMA protein in hippocampal tissue in the moxibustion group were decreased (P<0.05, P<0.01). CONCLUSION: Moxibustion could improve the cognitive function of VD rats, which may be related to reducing the expression of Bax, cytC, Tom20 and PUMA protein in hippocampal CA1 area, promoting the release of Bcl-2 and inhibiting the apoptosis of hippocampal neurons.

Mechanisms of Traditional Chinese Medicine Bushenantai granules in promoting angiogenesis at the maternal-fetal interface of recurrent spontaneous abortion mice.

OBJECTIVE: To assess the effects of Bushenantai (BSAT) granule() on angiogenesis-related factors [E2, P, and vascular endothelial growth factor (VEGF)] at the maternal-fetal interface of recurrent spontaneous abortion (RSA) mice, and to evaluate the role of BSAT in promoting angiogenesis at the maternal-fetal interface by influencing the expression of sex hormones, and VEGF. METHODS: A mouse model with normal pregnancy and another with Clark's classic RSA were established. The RSA mice were randomly assigned to six groups: normal, model, progesterone, high-doseBSAT granule (BSAT-H), medium-dose-BSAT granule (BSAT-M), and low-dose-BSAT granule (BSAT-L) (n = 10 for each group). The embryo loss rate and the histopathological changes in the decidual tissues were measured. Serum levels of estrogen (E2), progesterone (P), and VEGF were detected by enzyme-linked immunosorbent assay. The mRNA and protein expressions of estradiol receptor (ER), progesterone receptor (PR), VEGF, and vascular endothelial growth factor receptor 2 (VEGFR2) in the decidual tissues were identified by immunohistochemistry, Western blotting, and quantitative reverse transcription polymerase chain reaction. RESULTS: The embryo loss rate in all groups that received BSAT treatment was reduced, while the number of blood vessels at decidual tissues was increased. The serum levels of E2, P and VEGF were elevated, and the mRNA and protein expressions of ER, PR, VEGF, and VEGFR2 in the decidual tissues were enhanced. CONCLUSION: BSAT can improve angiogenesis at the maternal-fetal interface and reduce the embryo loss rate, which may be associated with its ability to increase the serum levels of estrogen, progesterone, and VEGF, in addition to up-regulation of mRNA and protein expression of ER, PR, VEGF, and VEGFR2 in the decidual tissue.

[Electroacupuncture improves learning-memory ability in diabetic rats with cognitive impairment via inhabitating proinflammatory cytokine production through p38 MAPK and STAT3 pathway].

OBJECTIVE: To investigate the effect of electroacupuncture (EA) on the p38 mitogen-activated protein kinase (p38 MAPK) and signal transducer and activator of transcription 3 (STAT3) pathway in hippocampus and frontal cortex of diabetic rats with cognitive impairment (CI), as well as the mechanism of EA in protection against CI in diabetic rats. METHODS: Thirty SD rats were divided into normal, model and EA groups (n=10 rats/group). The diabetic model was established by i.p.injection of Streptozotocin solution(25 mg/kg), followed by high-fat diet raising for 1 month, and the CI rats was confirmed by Morris water maze tasks. The rats in the EA group were given acupuncture at "Zusanli" (ST36) "Neiting" (ST44) and "Yishu" (EX-B3) 20 min/d, among which ST36 and ST44 were treated with EA. The treatment was conducted 6 times a week for 4 weeks. The fasting blood glucose (FBG) contents were assayed by glucometer before and after treatment. The rats' learning-memory ability was detected by Morris water maze tasks. The expression levels of IL-6、IL-1ß、TNF-α、p38 MAPK、p-p38 MAPK、STAT3 and p-STAT3 in hippocampus and frontal cortex were detected by Western blot and quantitative real-time PCR, separately. The mean fluorescence intensity of p38 MAPK and STAT3 was observed by immunofluorescence histochemistry. RESULTS: After modeling, FBG and the escape latency of Morris water maze tasks were significantly increased in the model group compared with the normal group (P<0.001, P<0.01). Following EA treatment, the increased FBG and average escape latency were markedly reversed in the EA group relevant to the model group (P<0.05). Compared with the normal group, the proteins and mRNAs expression of IL-6, IL-1ß, TNF-α, p38 MAPK, p-p38 MAPK, STAT3 and p-STAT3 in hippocampus and frontal cortex were significantly increased in the model group (P<0.001), as well as the mean fluorescence intensity of p38 MAPK and STAT3 in hippocampus and frontal cortex (P<0.001). Following EA intervention, the proteins and mRNAs expression of IL-6, IL-1ß, TNF-α, p38 MAPK, p-p38 MAPK, STAT3 and p-STAT3, and the mean fluorescence intensity of p38 MAPK and STAT3 in hippocampus and frontal cortex were down-regulated(P<0.001, P<0.05). CONCLUSION: EA can inhibit the over production of pro-inflammatory cytokines in diabetic rats with CI, possibly by regulating the expression of p38 MAPK and STAT3 pathway.

[Effect of moxibustion on learning-memory ability and expression of hippocampal inflammatory factors and microtubule associated proteins in vascular dementia rats].

OBJECTIVE: To observe the effect of moxibustion on learning-memory ability and expression of hippocampal inflammatory factors and microtubule-associated protein doublecortin (DCX, a marker of neuronal regeneration) in vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD. METHODS: SD rats were randomly divided into normal control, sham operation, VD model, moxibustion and medication groups (n=15 rats in each group). The VD model was established by repeated occlusion of the bilateral common carotid arteries and reperfusion. Moxibustion was applied to "Guanyuan" (CV4), "Mingmen" (GV4) and "Dazhui"(GV14) for 15 min, once a day, 6 days a week for 4 weeks. Rats of the medication group were treated by gavage of Nimodipine (2mg·kg-1·d-1) 3 times daily for 4 weeks. Morris water maze test was used to detect the average escape latency of location navigation tasks for assessing the rats' learning-memory ability. H.E. staining was used to detect histopathological changes of the hippocampus tissue. The number of DCX-positive neurons (DCX/NeuN co-expression) in the dentate gyrus (DG) region of hippocampus was counted under microscope after immunofluorescence double staining, the immunoactivity of hippocampal DCX detected by using immunohistochemistry stain and the expression of DCX, TNF-α, IL-1ß, MPO, NF-κB p65 and IL-6 proteins in the hippocampus tissue detected using Western blot. RESULTS: Following modeling, the average escape latency was significantly longer in the model group than in the normal control and sham operation groups (P<0.01), and notably shorter in both the moxibustion and medication groups than in the model group after the treatment (P<0.01, P<0.05). The number of DCX-positive neurons, and the expression levels of DCX, TNF-α, IL-1ß, MPO, NF-κB p65 and IL-6 proteins in the hippocampus were significantly increased in the model group in comparison with the normal control and sham operation groups (P<0.01, P<0.05). After the interventions and in comparison with the model group, the number of DCX-positive neurons and the expression level of DCX were further up-regulated in both moxibustion and medication groups (P<0.01), while the expression levels of hippocampal TNF-α, IL-1ß, MPO, NF-κB p65 and IL-6 proteins were considerably down-regulated in the moxibustion and medication groups (P<0.01). The effect of moxibustion was weaker than that of medication in down-regulating the expression of TNF-α,MPO, NF-κB p65, IL-6 and IL-1ß, and in up-regulating DCX-positive neuron number and DCX expression (P<0.05, P<0.01). H.E. staining showed loose arrangement of neurons (with vague neuronal membrane in some cells), uneven organelle chromatin, disappearance of partial nucleolus, necrocytosis, and infiltration of small number of lymphocytes after modeling, which was relatively milder in both moxibustion and medication groups. CONCLUSION: Moxibustion can improve learning-memory ability in VD rats, which may be related to its effect in down-regulating the expression of inflammatory factors and up-regulating the expression of DCX to promote neuronal repair and regeneration.

[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.

Does Moxa Smoke Have Significant Effect on the Acupuncturist's Respiratory System? A Population-Based Study.

OBJECTIVES: To evaluate the safety of moxa smoke, especially to provide quantitative information and details for the occupational prevention of acupuncturists. METHODS: We combined the questionnaire-based cross-sectional survey and lung function testing-based historical retrospective cohort research to investigate the safety of moxa smoke exposure (MSE) among acupuncturists. A mathematical regression model was established to quantitatively evaluate the relationship between moxa smoke exposure and the respiratory health of the acupuncturist. The smoke exposure time of the acupuncturist and the prevalence of abnormal respiratory symptoms or diseases were also evaluated. RESULTS: (1) The cross-sectional research showed that the incidence of expectoration (18.7%) and rhinitis (22.7%) was the most common respiratory symptom and disease after MSE. No statistical difference was found between smoke exposure time of the acupuncturist and the prevalence of abnormal respiratory symptoms or diseases, except the prevalence of rhinitis and shortness of breath (P < 0.01). Regression model for the incidence of first three symptoms (expectoration, shortness of breath, and wheezing) from the cross-sectional survey indicated that the weight coefficients of factors associated with moxa smoke were lower than those of factors unrelated to moxa smoke, such as gender and personal history of respiratory diseases. (2) Historical retrospective cohort research showed that there was no significant difference in the % predicted PEF. No statistic difference was found between the exposed and nonexposed group in large airway function indexes (% predicted FEV1, % predicted FVC, and % predicted FEV1/FVC) and small airway function indexes (% predicted FEF25, % predicted FEF50, % predicted FEF75, and % predicted MMEF), either. Especially, the % predicted MVV among males (106.23 ± 2.92 vs. 95.56 ± 1.92, P < 0.01 and % predicted VC among females (100.70 ± 1.59 vs. 95.91 ± 1.61, P < 0.05) between the two groups had statistical significance, but did not cause pulmonary ventilation dysfunction. CONCLUSIONS: MSE has no significant effect on the respiratory health of acupuncturists.

[Influence of acupuncture plus moxibustion on changes of synaptic structure and expression of synaptic skeleton related genes and proteins in prefrontal cortex of heroin re-addicted rats].

OBJECTIVE: To observe the effect of acupuncture plus moxibustion on the synaptic ultrastructure and expression of synaptic skeleton related proteins in the prefrontal cortex (PFC) of heroin re-addicted rats, so as to reveal its mechanisms underlying improvement of heroin addiction. METHODS: Twenty-four Wister rats (half male and half female) were randomly divi-ded into normal control, model and acupuncture groups (n＝8 in each group). The heroin re-addicted model was established by muscular injection of heroin into the hind limbs for 8 days (incremental 0.8-3.6 mg, once daily for 6 days, and twice daily for 2 days), followed by conventional breeding for 5 days (detoxification), the procedure (addition－detoxification) was repeated 3 cycles. For rats of the acupuncture group, "Baihui" (GV20) was needled with filiform needles which were retained for 30 min, and moxibustion was then applied to bilateral "Shenshu" (BL23) for 30 min. The treatment was conducted once daily during the deto-xification. On the 39th day of experiment, the bilateral prefrontal cortex tissues were sampled for examining the ultrastructure by using transmission electron microscope (TEM) after fixative solution immersion and for determining the expression of genes and proteins of activity-regulated cytoskeleton-associated protein (Arc), microtubule-asso-ciated protein-2 (MAP-2) and microtubule-associated protein Tau (Tau) with quantitative real-time PCR and Western blot, respectively. RESULTS: After modeling, the expression levels of Arc mRNA and protein were significantly up-regulated, and those of MAP-2 and Tau mRNA and proteins ob-viously down-regulated in the model group relevant to the normal control group (P<0.05). Following the intervention, the up-regulated Arc protein and mRNA and the down-regulated MAP-2 and Tau were obviously reversed relevant to the model group (P<0.05). Outcomes of TEM showed unclear pre- and post-membranes of the synapses, narrowing of the synaptic gap and non-uniform of the density of the thickened dense plaque after modeling, which was relatively milder in the acupuncture group. CONCLUSION: Acupuncture plus moxibustion can improve changes of synaptic ultrastructure in heroin re-addicted rats, which may be related to their effect in regulating the expression of some synaptic skeleton proteins and genes.

[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.

[Effect of Different Concentrations of Moxa-smoke on Lung Function and TNF-α and IL-1 ß Levels in Serum and Lung Tissues in Normal Rats].

OBJECTIVE: To study the effect of moxa-smoke inhaling on the respiratory system, so as to provide experimental data and theoretical basis for evaluating the safety of moxa-smoke inhaling during moxibustion treatment. METHODS: A total of 48 SD rats were randomized into control, low, medium and high moxa-smoke-concentration groups (n＝12 in each group). The low, medium and high concentrations of smoke were controlled in (0.11±0.05) mg/m3, (0.23±0.05) mg/m3 and (0.53±0.05) mg/m3 respectively in each of 3 glass boxes (with reference to the level of PM 2.5). The smoking was conducted 4 hours each time, twice a day for 100 days. The normal group did not receive any moxa-smoke inhaling. The histopathological changes of lung and bronchial tissues were detected by H．E. stainning, and the contents of TNF-α and IL-1 ß of plasma, bronchoalveolar la-vage fluid (BALF) and lung tissue detected by ELISA. The levels of forced vital capacity (FVC), forced expiratory volume (FEV), FEV 0.3/FVC (0.3＝ the 0.3rd second), maximal mid-expiratory flow rate(MMEF), peak expiratory flow(PEF) were detected by animal pulmonary function analysis system. RESULTS: After 100 days' moxa-smoke inhaling, the contents of TNF-α in the plasma, BALF and lung tissues and IL-1 ß in the lung tissue of the low, medium and high concentration moxa-smoke groups, and IL-1 ß in the plasma and BALF of the medium and high concentration groups were significantly increased relevant to the control group (P<0.05, P<0.01). H．E. stain showed various inflammatory changes in the lungs and trachea tissues, including obvious fusion of pulmonary alveoli, lymphocyte infiltration, increase of capillary permeability, red blood cell exudation, etc. in the high concentration group, these situations were milder in the medium concentration group and were not obvious in the low concentration group. Compared with the control group, there were no significant changes in the FVC, FEV, FEV 0.3/FVC, MMEF and PEF of lung function in the three concentration groups (P> 0.05)．. CONCLUSION: Long term inhalation of high concentration of moxa-smoke may lead to inflammatory injury in the lung and bronchial tissues but has no significant effect on the respiratory function in rats. Nevertheless, a good air-ventilation during moxibustion in a treatment room is necessary.

[Moxibustion Improves Learning Ability by Regulating Hippocampal Neurotrophic Factor Expression and Notch Signaling in Vascular Dementia Rats].

OBJECTIVE: To observe the effect of moxibustion on the learning ability and expression of neurotrophic factors and Notch signaling in vascular dementia (VD) rats, so as to explore its neurogenesis mechanism underlying improvement of VD. METHODS: Sixty SD rats were equally and randomly divided into sham operation (sham), model, medication and moxibustion groups (n=15 rats/group). The VD model was established by occlusion of the bilateral cervical common arteries and reperfusion. Moxibustion was applied to "Dazhui"(GV 14), "Guanyuan"(CV 4) and "Mingmen"(GV 4)for 15 minutes, once daily, 6 times a week for 4 weeks. Rats of the medication group were treated by gavage of nimodipine (2 mg·kg-1·d-1),3 times a day, 6 days a week for 4 weeks. Morris water maze tests were performed to detect the rat's learning-memory ability. The infarcted size of the brain was detected by using 2,3,5-triphenyltetrazolium chloride (TTC) staining, and H.E. staining was used to detect the histopathological changes. The expression level of glia fibrillary acidic protein (GFAP), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), Notch 1 (a receptor), Hes 3 (a downstream effector) mRNAs and proteins in the hippocampal tissues were detected by quantitative real-time PCR and Western blot, respectively. RESULTS: After 4 weeks' intervention, modeling-induced increase of escape latency was significantly shortened in both moxibustion and medication groups relevant to the model group (P<0.05), and the infarct size was reduced and the damage degree of nerve cells in the brain tissue alleviated. The expression levels of BDNF, NGF, GFAP, Hes 3, Notch 1 genes and proteins were significantly up-regulated in the model group relevant to the sham operation group (P<0.05,P<0.01). After the intervention, the expression levels of hippocampal BDNF, NGF, GFAP, Hes 3 and Notch 1 mRNAs and proteins in the moxibustion group, and NGF and GFAP mRNAs, and BDNF, NGF, GFAP, Hes 3 and Notch 1 proteins in the medication group were further obviously up-regulated relevant to the model group (P<0.05, P<0.01). The effect of moxibustion was significantly superior to that of medication in up-regulating GFAP, Hes 3, Notch 1 mRNAs expression (P<0.05,P<0.01). No significant differences were found in the expression of BDNF, Hes 3 and Notch 1 mRNAs in the medication group relevant to the model group (P<0.05), and between the moxibustion and medication groups in up-regulating the expression of BDNF and NGF mRNAs, and in up-regulating the expression of BDNF, NGF, GFAP, Hes 3 and Notch 1 proteins (P<0.05). CONCLUSIONS: Moxibustion can improve learning ability in VD rats, which may be associated with its effects in up-regulating the expression of neurotrophic factors and in potentiating Notch signaling.

[Protective Effect of Acupuncture Serum Derived from Acute Convulsion Rats on Cultured Hip-pocampal Neurons with Seizure-like Discharges by Regulating Expression of Endoplasmic Reticulum Stress-inducible Molecular Chaperones].

OBJECTIVE: To observe the effect of acupuncture serum on the number of apoptosis of the cultured hippocampal neurons with seizure-like discharges and the expression levels of endoplasmic reticulum stress-inducible molecular chaperones glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP) and cysteine protease protein-12(Caspase-12), so as to reveal its protective mechanism on seizure-induced injury of hippocampal neurons. METHODS: A regular primary culture of neurons derived from the hippocampus of the newly-born SD rats was conducted for 10 days, then these cultured neurons that displayed seizure-like discharges in Mg2+-free medium were divided into normal extracellular fluid (medium) group (normal), Mg2+-free medium group, acupuncture serum group and non-acupuncture serum group (n=30). Blood examples were taken from pentylenetetrazol (i.p.i.) -induced acute convulsion adult SD rats underwent manual acupuncture stimulation of "Baihui" (GV 20) and "Dazhui" (GV 14, once daily for 7 days) to prepare acupuncture serum. Hippocampal neuronal cultures were prepared from hip-pocampal tissue isolated from 24 h-old SD rats. The isolated neurons were incubated normally in Dulbecco's Modified Eagle Me-dium (DMEM)/Nutrient Mixture F 12(1:1) for 10 days, followed by exposure to the extracellular fluid {composed of NaCl, KCl, CaCl2, MgCl2, HEPES[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], D-glucose and aminoethanoic acid (pH 7.3)} for 3 h and returned to normal culture for normal group, by exposure to Mg2+-free extracellular fluid (to induce seizure-like discharges) for 3 h and returned to normal culture for Mg2+-free medium group, by exposure to Mg2+-free extracellular fluid for 3 h and returned to a regular culture medium[DMEM/F 12(1:1)] plus acupuncture serum (9:1) for acupuncture serum group, and by exposure to Mg2+-free extracellular fluid for 3 h and returned to DMEM/F 12(1:1) plus non-acupuncture serum (9:1) for non-acupuncture serum group. The cell apoptosis was assessed at 2, 12 and 48 h after application of acupuncture serum or non-acupuncture serum by using TUNEL method and the expression levels of GRP 78, CHOP and Caspase-12 proteins of the cultured cells at the 3 time-points detected using Western blot. RESULTS: The number of apoptotic hippocampal neurons was significantly higher in the Mg2+-free medium group than in the normal medium group at 2, 12 and 48 h (P<0.01), and considerably decreased in the acupuncture serum group (but not in the non-acupuncture serum group) relevant to the Mg2+-free medium group at the same time-points after application of acupuncture serum (P<0.05, P<0.01). The expression levels of GRP 78 protein at 2 h, CHOP and Caspase-12 proteins at 2, 12 and 48 h were significantly up-regulated in the Mg2+-free medium group relavant to the normal me-dium group (P<0.05, P<0.01). After application of acupuncture serum to the culture medium, the expression levels of GRP 78 protein at the three time-points were significantly increased in comparison with the Mg2+-free medium group (P<0.01,P<0.05), while those of CHOP at 12 and 48 h, and Caspase-12 at the three time-points were notably down-regulated (P<0.05,P<0.01). No significant differences were found between the non-acupuncture serum and Mg2+-free medium groups in the expression levels of GRP 78, CHOP and Caspase-12 proteins at the three time-points (P>0.05). CONCLUSIONS: Acupuncture serum can significantly reduce apoptosis of the cultured hippocampal neurons, which may be related to its effects in increasing the expression of GRP 78 protein and down-regulating the expression of CHOP and Caspase-12 proteins, suggesting an important role of acupuncture serum in maintaining the stability of the neuronal endoplasmic reticulum.

Atlantoaxial Misalignment Causes High Blood Pressure in Rats: A Novel Hypertension Model.

Atlantoaxial disorders are often correlated with hypertension in practice. In order to study the relationship between atlantoaxial disorder and hypertension, we attempted to construct an animal model. In this work, we presented an animal model where their atlantoaxial joints were misaligned. We investigated the changes of blood pressure before and after treatments of the modeled rats. We had the following results. (1) SBP and DBP of each surgery group were significantly higher than those of control and sham groups. (2) After the second operation (the fixture was removed), SBP and DBP of both surgery groups decreased and got closer to the control and sham groups after 7 days. (3) Heart rates got significantly higher in both surgery groups, compared to control and sham groups. (4) The blood Ach levels of the surgery groups were significantly lower than those of control and sham groups. With these results, we concluded that we successfully constructed cervical atlantoaxial disorder models in rats that showed hypertension symptom. However, the underlying mechanism connecting atlantoaxial disorder and hypertension still requires further study.

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.

Acupuncture regulates the unfolded protein response and inhibits apoptosis in a rat model of heroin relapse.

OBJECT: To explore the unfolded protein response (UPR) in the hippocampus of rats undergoing heroin relapse and the mechanisms underlying the acupuncture-mediated inhibition of brain damage caused by heroin relapse. METHODS: 60 Sprague-Dawley rats (30 females and 30 males) were randomly divided into four groups: Control group, Heroin group, Heroin+acupuncture group, and Heroin+methadone group (n=15 each). In the latter three groups, a model of heroin addiction was established by successive increments of intramuscular heroin injections for 8 days, according to the exposure (addiction)→detoxification method. A UPR RT2 Profiler PCR array was used to screen for differentially expressed genes in the hippocampus. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. The protein expression levels of the following three differentially expressed genes were detected by Western blot to validate the results of the PCR array: heat shock protein (HSP)70, HSP105, and valosin-containing protein (Vcp). RESULTS: The UPR RT2 Profiler PCR Array detection results indicated that acupuncture increased the expression levels of the molecular chaperones HSP70, HSP105, and Vcp. The degree of neuronal apoptosis in the hippocampus of rats in the Heroin+acupuncture and Heroin+methadone groups was significantly reduced compared with the untreated Heroin group (p<0.01). Protein expression of HSP70, HSP105, and Vcp in the Heroin+acupuncture and Heroin+methadone groups was significantly higher than the Heroin group (p<0.01). CONCLUSIONS: The positive effects of acupuncture on brain damage caused by heroin may be closely related to up-regulation of HSP70, HSP105, and Vcp, and reduced apoptosis.