Combination of bone marrow mesenchymal stem cells and moxibustion restores cyclophosphamide-induced premature ovarian insufficiency by improving mitochondrial function and regulating mitophagy.

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.

[Electroacupuncture promotes expression of glutathione related regulatory enzymes in ovary tissue of rats with diminished ovarian reserve].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on ovarian function and expression of glutathione (GSH) related regulatory enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione reductase (GR) protein and gene in rats with diminished ovarian reserve (DOR), so as to explore its mechanisms underlying up-regulation of antioxidant stress ability. METHODS: A total of 30 female SD rats with normal estrous cycle were randomly divided into blank control, model and EA groups, with 10 rats in each group. The DOR model was established by gavage of tripterygium wilfordii polyglycoside suspension (50 mg·kg-1·d-1) for 14 consecutive days, while the rats in the blank group were given equal volume of 0.9% sodium chloride solution. One hour after daily gavage, EA (1.0 mA, 100 Hz) was applied alternately to bilateral "Shenshu"(BL23), and "Zhongwan"(CV12)+"Guanyuan"(CV4) for 10 min, for 14 consecutive days. Estrous cycles of rats in each group were observed and recorded daily during intervention.After the intervention, H.E.staining was used to observe histopathological changes of the ovarian tissue. The contents of serum sex hormones ï¼»follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), estradiol (E2)ï¼½ and oxidative damage markers ï¼»8-hydroxydeoxyguanosine (8-OHDG) and nitrotyrosine (NTY)ï¼½ were determined by ELISA. The contents of GSH and oxidized glutathione (GSSG) in the liver tissue were determined by colorimetry, and their ratios were calculated. Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the immunoactivity and gene expression levels of γ-GCS and GR in the ovarian tissues, respectively. RESULTS: Compared with the blank group, the model group had a marked increase in the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents (P<0.01) and a considerable decrease in the levels of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number as well as the expression of γ-GCS and GR mRNAs (P<0.05, P<0.01). After EA intervention, the increase of the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents and the decrease of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number of γ-GCS and GR as well as the expression of γ-GCS genes were all reversed (P<0.01, P<0.05). H.E. staining showed degenerative changes of the ovarian tissue, fewer follicles at every level and increase of atretic follicles, disarrangement and layer number decrease of granulosa cells, and atrophy of corpus luteum in the model group, which were relatively milder in the EA group. CONCLUSION: EA can improve ovarian function, and reduce oxidative stress damage in DOR rats, which may be associated with its functions in up-regulating the expression of γ-GCS and GR protein and gene in the ovarian tissue.

[Protective effect of moxibustion preconditioning in rats with premature ovarian insufficiency].

OBJECTIVE: To observe the effects of moxibustion preconditioning on ovarian function, fertility and ovarian granulosa cell apoptosis in rats with premature ovarian insufficiency (POI), so as to investigate its underlying mechanism in improving POI. METHODS: Forty-two female SD rats with two complete estrous cycles were randomly divided into control group, model group and pre-moxibustion group, with 14 rats in each group. The pre-moxibustion group was pretreated with mild moxibustion for 14 days before POI model establishment at 1) "Guanyuan" (CV4) and "Zhongwan" (CV12) and 2) bilateral "Shenshu" (BL23) as two sets of acupoints on alternate days, once each day, for 10 min each acupoint. After 14-day mild moxibustion intervention, 75 mg·kg-1·d-1 tripterygium glycoside tablet suspension was administered to rats in the pre-moxibustion group and the model group by gavage, for 14 consecutive days, while equivalent saline was given to rats in the control group in the same way. After modeling, the effect of moxibustion preconditioning on ovarian reserve function was evaluated by the estrous cycles, pregnancy rate and embryo number, morphological changes of ovaries, and serum sex hormone levels. TUNEL staining was used to detect the rate of granulosa cell apoptosis in ovaries. Immunohistochemistry and real time quantitative PCR were used to detect the relative expression of Caspase-3 and Caspase-9 proteins and mRNA levels in ovaries. RESULTS: Compared with the control group, the estrous cycles were disturbed; the pregnancy rate and number of embryos, the wet weight of ovary and ovarian index, the number of total follicles and different level of follicles, serum Estradiol (E2) and anti-mullerian hormone (AMH) levels were all significantly decreased (P<0.01,P<0.05), while the number of atretic follicles, serum follicule-stimulating hormone (FSH) and luteinizing hormone (LH) levels, the number of TUNEL-positive granulosa cells, the expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were significantly increased (P<0.01) in the model group. Compared with the model group, the disordered estrous cycles were improved; the pregnancy rate, the embryo numbers, the wet weight of ovary, and the total follicle number and primary follicle number, serum AMH level were significantly increased (P<0.01，P<0.05), while the number of atretic follicles, serum FSH level, the number of TUNEL-positive granulosa cells, expression of ovarian Caspase-3 and Caspase-9 proteins and mRNAs were all significantly decreased (P<0.01,P<0.05) in the moxibustion group. CONCLUSION: Moxibustion preconditioning could improve ovarian function and improve fertility of POI rats, which may be associated with reducing the apoptosis of ovarian granulosa cells.

Effects of acupuncture treatment on microRNAs expression in ovarian tissues from Tripterygium glycoside-induced diminished ovarian reserve rats.

Acupuncture is widely used to improve ovarian function. Previously, we demonstrated that acupuncture can improve oxidative stress in rats with tripterygium glycoside tablet suspension (TG)-induced diminished ovarian reserve (DOR). Herein, we aimed to explore the antioxidation mechanism of acupuncture for ameliorating the ovarian reserve in DOR rats. We performed microRNA sequencing and bioinformatics analysis to screen differentially expressed miRNAs (DE miRNAs) in ovarian tissues. In total, 1,172 miRNAs were identified by miRNA sequencing, of which 28 DE miRNAs were detected (including 14 upregulated and 14 downregulated) in ovarian tissues from the acupuncture group when compared with the DOR model rats. Based on functional enrichment analysis, the target genes of DE miRNAs were significantly enriched in GO-biological process (BP) terms associated with biological processes, positive regulation of transcription by RNA polymerase II, signal transduction, regulation of transcription, DNA-templated processes, and oxidation-reduction processes. In the Kyoto Encyclopedia of Genes and Genomes analysis, the main pathways were the MAPK signaling pathway, hepatitis B, proteoglycans in cancer, human cytomegalovirus infection, and the Ras signaling pathway. Finally, reverse transcription-quantitative PCR results confirmed that rno-miR-92b-3p, mdo-miR-26b-5p_R+1_1ss10TC, and bta-miR-7857-3p_R-1 were downregulated in the acupuncture group. The results revealed the impact of acupuncture on miRNA profiling of ovarian tissues from DOR rats, suggesting that rno-miR-92b-3p, mdo-miR-26b-5p_R+1_1ss10TC, and bta-miR-7857-3p_R-1 might provide relevant cues to relieve DOR-mediated oxidative stress.

Moxibustion alleviates decreased ovarian reserve in rats by restoring the PI3K/AKT signaling pathway.

OBJECTIVE: Moxibustion, a common therapy in traditional Chinese medicine, has potential benefits for treating decreased ovarian reserve (DOR). The present study investigates the protective effect of moxibustion in a rat model of DOR and explores the possible mechanisms. METHODS: Sixty-four female Sprague-Dawley rats were randomly divided into four groups: control, DOR, moxibustion (MOX), and hormone replacement therapy (HRT). The DOR rat model was established by intragastric administration of 50 mg/kg Tripterygium glycoside suspension (TGS), once daily for 14 days. MOX and HRT treatments were given from the day TGS administration was initiated. The ovarian reserve function was evaluated by monitoring the estrus cycle, morphological changes in ovaries, levels of serum estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and anti-Mullerian hormone (AMH), pregnancy rate and embryo numbers. Terminal-deoxynucleotidyl transferase-mediated nick-end-labeling staining was used to identify ovarian granulosa cell apoptosis, while the protein and mRNA expressions of Bax, B-cell lymphoma-2 (Bcl-2), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in ovarian tissues were examined by immunohistochemistry, Western blot and quantitative reverse transcription-polymerase chain reaction. RESULTS: Compared with the DOR group, MOX improved the disordered estrous cycle, promoted follicular growth, reduced the number of atresia follicles, increased the concentrations of serum E2 and AMH, and decreased serum FSH and LH concentrations. More importantly, the pregnancy rate and embryo numbers in DOR rats were both upregulated in the MOX treatment group, compared to the untreated DOR model. Further, we found that the MOX group had reduced apoptosis of ovarian granulosa cells, increased Bcl-2 expression and reduced expression of Bax. Furthermore, the PI3K/AKT signaling pathway was triggered by the moxibustion treatment. CONCLUSION: Moxibustion improved ovarian function and suppressed apoptosis of ovarian granulosa cells in a rat model of DOR induced by TGS, and the mechanism may involve the PI3K/AKT signaling pathway.

Moxibustion Ameliorates Ovarian Reserve in Rats by Mediating Nrf2/HO-1/NLRP3 Anti-Inflammatory Pathway.

Diminished ovarian reserve (DOR) is an increasingly emerging reproductive disorder that disturbs reproductive-aged women, which is closely linked with inflammation. In clinic, moxibustion has already been applied for reproductive problems. In the present study, we examined the involvement of inflammation in DOR and investigated the effect of moxibustion for its anti-inflammatory activities. Methods. DOR rat model was established using tripterygium glycosides A tablets (TGs) suspension by intragastric administration and was then treated with either moxibustion or hormone replacement therapy (HRT), respectively. Estrus cycles were observed through vaginal cytology. Ovarian morphological alterations were observed by HE staining. The serum levels of follicle-stimulating hormone (FSH), estradiol (E 2), anti-Müllerian hormone (AMH), tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) were measured through ELISA. The expression levels of Nrf2, HO-1, and NLRP3 were detected using immunohistochemistry. Nrf2, HO-1, and NLRP3 mRNA were examined by RT-PCR. Results. Moxibustion improved estrus cycles, FSH, E 2, and AMH levels relative to DOR rats as well as HRT, while also inhibiting ovarian tissue injury. Anti-inflammatory cytokine IL-10 in peripheral blood was upregulated, and proinflammatory factor TNF-α was decreased after treatment with moxibustion. Moxibustion enhanced the expression of mRNA and protein of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1); in the mean time, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) was suppressed. Conclusions. We demonstrated that moxibustion could ameliorate the ovarian reserve in rats induced by TGs. Overall, the effect of moxibustion was comparable to that of HRT. The underlying mechanism could be attributed to the anti-inflammatory effects of moxibustion, which suppressed NLRP3 activation by upregulating Nrf2/HO-1 signaling pathway.

[Effect of moxibustion on Nrf2/HO-1 signaling pathway in rats with diminished ovarian reserve].

OBJECTIVE: To observe the effect of moxibustion on Nrf2/HO-1 signaling pathway in rats with diminished ovarian reserve (DOR), and to explore the protective mechanism of moxibustion on ovarian reserve function. METHODS: Forty SD rats were randomly divided into a blank group, a model group, a moxibustion group and a hormone group, 10 rats in each group. The rats in the model group, moxibustion group and hormone group were treated with intragastric administration of tripterysium glycosides turbid liquid to prepare DOR model. The rats in the blank group were treated with intragastric administration of sodium chloride solution with the same volume, once a day for 14 days. The rats in the hormone group were treated with hormone sequential therapy for 14 days from the day of modeling; the rats in the moxibustion group were treated with moxibustion at bilateral "Shenshu" (BL 23) or "Guanyuan" (CV 4) and "Zhongwan" (CV 12) from the day of modeling, and the two groups acupoints were alternated every other day, 10 min each time, for 14 consecutive days. The estrus cycle was observed every day by vaginal exfoliated cell smear, and the estrus cycle disorder rate in each group was calculated. After the intervention, the HE staining was used to observe the histological morphology of ovaries; ELISA was used to detect the contents of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), anti Mullerian hormone (AMH), superoxide dismutase (SOD) and malondialdehyde (MDA); the protein levels of Nrf2 and HO-1 in ovarian tissue were detected by immunohistochemistry; real-time PCR (TaqMan probe method) was used to detect the expression of Nrf2 and HO-1 mRNA. RESULTS: Compared with the blank group, the rate of estrus cycle disorder in the model group was increased (P<0.01); compared with the model group, the rate of estrus cycle disorder in the moxibustion group and hormone group was decreased (P<0.01). Compared with the blank group, the serum contents of FSH, LH and MDA in the model group were increased (P<0.01), and the serum contents of E2, AMH and SOD were decreased (P<0.01). Compared with the model group, the serum contents of FSH, LH and MDA in the moxibustion group and hormone group were decreased (P<0.01, P<0.05), and the serum contents of E2, AMH and SOD were increased (P<0.01). Compared with the blank group, the protein and mRNA expression of Nrf2 and HO-1 in the model group were decreased (P<0.01); compared with the model group, the protein and mRNA expressions of Nrf2 and HO-1 in the moxibustion group and hormone group were increased (P<0.01). CONCLUSION: Moxibustion could reduce the rate of estrus cycle disorder, improve the level of serum sex hormones and antioxidant stress in DOR rats, and the mechanism may be related to the regulation of Nrf2/HO-1 signaling pathway.

[Effect of acupuncture on oxidative stress and apoptosis in ovarian hypofunction induced by Tripterygium Glycosides].

OBJECTIVE: To explore whether manual acupuncture stimulation of "Shenshu " (BL23), "Guanyuan" (CV4), "Zhongwan" (CV12) can improve the ovarian function by resisting oxidant stress and reducing apoptosis of granulosa cells in rats with ovarian hypofunction. METHODS: Forty female SD rats with normal estrous cycles were randomly divided into blank control, model, hormone therapy and acupuncture groups (n=10 rats in each group). The ovarian hypofunction model was established by gavage of Tripterygium Glycosides suspension (50 mg·kg-1·d-1) for 14 successive days. Rats of the hormone therapy group were treated by gavage of estrogen and progesterone, and those of the acupuncture group treated by manual acupuncture stimulation of bilateral BL23 or CV4 and CV12 alternatively by using uniform reinforcing-reducing method for 10 s every 5 min (3 times in 10 min). The treatment was performed once daily for 14 days. The blank group was given equal volume of normal saline daily. On the 9th day, the estrous cycle of each rat was observed by means of vaginal smear test. The ovarian index, serum estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), malondialdehyde (MDA) and superoxide dismutase (SOD) contents were detected by ELISA. Histopathological changes of the ovary tissue were observed by H.E. staining. The expressions of apoptosis-related proteins Bax and Bcl-2 and their mRNAs of the ovaries were determined by immunohistochemical staining and quantitative real time-PCR separately. RESULTS: The menstrual disorder rate was 100% in the model group, and was significantly higher than those in the hormone therapy (30%) and acupuncture (40%) groups (P<0.01). Following modeling, the ovarian wet weight and index, E2 and SOD contents, Bcl-2 protein and mRNA expressions were significantly decreased (P<0.01, P<0.05), and serum FSH, LH and MDA levels, Bax protein and mRNA expressions were significantly increased (P<0.05, P<0.01) in the model group in comparison with the blank control group. H.E. staining showed a large amount of connective tissue in the ovary, with fewer mature follicles, increase of atresia follicles, significant reduction of luteal tissue, and appearance of scarring tissue in the model group. Compared with the model group, there were more mature follicles, fewer atresia follicles, lower abnormal granulosa cell morphology and lower estrous cycle disorder ratio in both hormone therapy and acupuncture groups. After the interventions, the decreased ovarian wet weight and index, serum E2 and SOD contents, expressions of Bcl-2 protein and mRNA were significantly increased (P<0.05, P<0.01), and the increased levels of FSH and LH, MDA, expressions of Bax protein and mRNA were significantly decreased in both hormone therapy and acupuncture groups (P<0.05, P<0.01). The effect of acupuncture was significantly superior to that of hormone in up-regulating SOD, Bax and Bcl-2 protein expressions (P<0.01). CONCLUSION: Acupuncture can improve the menstrual disorder in rats with ovarian hypofunction, which is closely related to its effects in improving antioxidant stress ability and regulating the expressions of apoptosis-related protein and mRNA of ovarian gra-nulosa cells.

Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway.

Electroacupuncture (EA) has anti-oxidative and anti-inflammatory actions, but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion (I/R) injury involves modulation of the extracellular regulated kinase 1/2 (ERK1/2) signaling pathway is unclear. Middle cerebral artery occlusion (MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours. A 30-minute period of EA stimulation was applied to both Baihui (DU20) and Dazhui (DU14) acupoints in each rat (10 mm EA penetration depth, continuous wave with a frequency of 3 Hz, and a current intensity of 1-3 mA) when reperfusion was initiated. EA significantly reduced infarct volume, alleviated neuronal injury, and improved neurological function in rats with MCAO. Furthermore, high mRNA expression of Bax and low mRNA expression of Bcl-2 induced by MCAO was prevented by EA. EA substantially restored total glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels. Additionally, Nrf2 and glutamylcysteine synthetase (GCS) expression levels were markedly increased by EA. Interestingly, the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059 (a specific MEK inhibitor). Collectively, our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA. Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA.

Electroacupuncture Attenuates Cerebral Ischemia and Reperfusion Injury in Middle Cerebral Artery Occlusion of Rat via Modulation of Apoptosis, Inflammation, Oxidative Stress, and Excitotoxicity.

Electroacupuncture (EA) has several properties such as antioxidant, antiapoptosis, and anti-inflammatory properties. The current study was to investigate the effects of EA on the prevention and treatment of cerebral ischemia-reperfusion (I/R) injury and to elucidate possible molecular mechanisms. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 24 h. EA stimulation was applied to both Baihui and Dazhui acupoints for 30 min in each rat per day for 5 successive days before MCAO (pretreatment) or when the reperfusion was initiated (treatment). Neurologic deficit scores, infarction volumes, brain water content, and neuronal apoptosis were evaluated. The expressions of related inflammatory cytokines, apoptotic molecules, antioxidant systems, and excitotoxic receptors in the brain were also investigated. Results showed that both EA pretreatment and treatment significantly reduced infarct volumes, decreased brain water content, and alleviated neuronal injury in MCAO rats. Notably, EA exerts neuroprotection against I/R injury through improving neurological function, attenuating the inflammation cytokines, upregulating antioxidant systems, and reducing the excitotoxicity. This study provides a better understanding of the molecular mechanism underlying the traditional use of EA.

[Construction of inheritance way of acupuncture and moxibustion science based on tacit knowledge].

The conception and characteristics of tacit knowledge and the tacit knowledge in the science of acupuncture-moxibustion are analyzed in this paper. It is proposed that the attention should be paid to digging the tacit knowledge in the science of acupuncture-moxibustion and constructing the corresponding inheritance way, which could effectively improve the students' cultivation quality and reach the aim of talent cultivation centered on the clinical thinking ability, acupuncture operation skill and clinical innovation ability.

[Effect of electroacupuncture on expression of gamma-glutamylcysteine synthetase protein and mRNA in cerebral cortex in rats with focal cerebral ischemia-reperfusion].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Baihui" (GV 20) and "Dazhui" (GV 14) on the expression of gamma-glutamylcysteine synthetase (gamma-GCS) protein and gene in the cerebral cortex in rats with cerebral ischemia-reperfusion (CI/R) injury, so as to explore its molecular biological mechanism underlying anti-oxidative stress. METHODS: A total of 30 male Sprague-Dawley rats were randomized into sham operation (sham, n = 10), model (n = 10), and EA (n = 10) groups. CI/R model was established by right middle cerebral artery occlusion (modified Longa's thread occlusion method) for 2 hours and reperfusion for 24 hours. EA (3 Hz, 1-3 mA) was applied to "Dazhui" (GV 14) and "Baihui" (GV 20) for 30 min. gamma-GCS protein expression of the parietotemporal region of cerebral cortex was detected by immunohistochemistry and gamma-GCS heavy subunit (gamma-GCSh) mRNA and gamma-GCS light subunit (gamma-GCSI) mRNA expression levels were assayed by real-time quantitative polymerase chain reaction (RT-PCR). RESULTS: Compared with the sham group, the expression levels of gamma-GCS protein in the pyramidal cell layer of the cerebral cortical parietotemporal region, and y-GCSh mRNA and gamma-GCSI mRNA, and the number of gamma-GCS immuno-reaction positive cells had no remarkable changes in the model group (P > 0.05), while in comparison with the model group, the expression levels of cerebral cortical gamma-GCS protein, and gamma-GCSh mRNA and gamma-GCSI mRNA, and the number of gamma-GCS immuno-reaction positive cells were increased considerably in the EA group (P < 0.01, P < 0.05). CONCLUSION: EA of GV 20 and GV 14 can upregulate expression levels of gamma-GCS protein, gamma-GCSh mRNA and gamma-GCSI mRNA of the cerebral cortical parietotemporal region in CI/R rats, which may contribute to its effect in protecting cerebral cortical cells from injury by clearing away excessive oxygen free radicals.

[Effects of electroacupuncture on expression of calmodulin in the hippocampus of rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To investigate the protective effect of electroacupuncture (EA) on injured neurons and the signal transduction mechanism of calmodulin (CaM) in rats with cerebral ischemia-reperfusion injury (CIRI). METHODS: A total of 25 SD rats were randomly divided into a sham-operation group, a model group, an EA. group, a TFP group and an EA+TFP group. The rat model of middle cerebral artery occlusion (MCAO) was established by the modified Longa thread occlusion method. The EA group was treated with EA at "Dazhui" (GV 14) and "Baihui" (GV 20) for 30 minutes. The TFP group was treated with lumbar intrathecal injection of Trinuoperazine (TFP) at a dose of 40 microL/kg, the inhibitor of CaM. The EA + TFP group was treated with EA combined with TFP, and the sham-operation group and the model group without any treatment. The neurology deficit score was evaluated by the Julio's neuroethology score methods in all rats, and the expression of CaM in cerebral hippocampus tissue was detected with immunohistochemical method in different intervention condition. RESULTS: (1) In comparison with the model group of 6.90 +/- 1.66, the neuroethology score in the EA group of 14.50 +/- 1.08, the TFP group of 11.70 +/- 1.06 and the EA + TFP group of 14.30 +/- 1.06 were all significantly increased (all P < 0.01), while those still were all lower than the sham group of 17.60 +/- 0.52 (all P < 0.01), and the EA group was better than the TFP group (P < 0.01). (2) In comparison with the sham group of 0.080 +/- 0.045, the immune positive expression score of CaM protein in hippocampus in the model group of 1.680 +/- 0.268 was sig nificantly increased (P < 0.01). In comparison with the model group, the expression score of CaM protein in the EA group of 0.880 +/- 0.179, the TFP group of 0.720 +/- 0.179 and the EA + TFP group of 0.420 +/- 0.249 were all significantly reduced (all P < 0.01), and the expression score of CaM in the EA + TFP group was lower than that in the TFP group (P < 0.05). CONCLUSION: EA can reduce the injury of cerebral neurons induced by CIRI in rats and promote the recovery, which may be related to its effect in regulating CaM signaling pathway after the ischemia-reperfusion injury.

[Effect of electroacupuncture on hippocampal LTP in Alzheimer' s disease rats induced by Abeta(25-35)].

OBJECTIVE: To observe the effect of electroacupuncture(EA) on the learning-memory ability in Alzheimer's disease(AD) rats. METHODS: Thirty SD rats were equally randomized into control, model and EA groups. AD model was established by injecting beta-amyloid (Abeta(25-35), 10 microg) into the bilateral dentate gyri of the hippocampal CA 1 area (AP--3.5 mm, ML +/- 2.0 mm, DV 2.7 mm). EA (4 Hz, 1-2 mA) was applied to "Baihui" (GV 20), "Dazhui" (GV 14), bilateral "Shenshu" (BL 23) and bilateral "Yongquan" (KI 1) for 30 min, once daily for 7 days. The learning-memory ability was detected by using step-down test. Long term potentiation (LTP) of hippocampal CA 1 area was recorded by using tungsten microelectrodes after high frequency stimulation (HFS) conditioning of the cortical anterior perforated substance. RESULTS: In AD rats, the error number and total error time of step-down test were increased significantly (P < 0.05, P < 0.01), and the slope of the excitatory postsynaptic potential (EPSP), and the amplitude and area of population spikes (PS) at 30 min, 60 min, 90 min and 120 min after high-frequency stimulation of the cortical anterior perforated substance lowered markedly in comparison with sham operation (control) group (P < 0.05, P < 0.01). After EA treatment, the error number and total error time decreased significantly, the slope of EPSP, and the amplitude and area of PS from 30 min to 120 min after high-frequency stimulation increased considerably in comparison with model group (P < 0.05, P < 0.01), suggesting an improvement of the synaptic transmission after EA. CONCLUSION: EA can improve AD rats' learning-memory ability and raise the slope of EPSP and the amplitude of PS, which may contribute to its effect in relieve symptoms of AD in clinic.

[Effect of electroacupuncture on cerebral cortex ultrastructure in rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Baihui" (GV 20) and "Dazhui" (GV 14) on the ultrastructure of cerebral cortex in rats with cerebral ischemia reperfusion (CI/R) injury. METHODS: Fifteen SD rats were equally randomized into sham-operation (sham), model and EA groups. CI/R model was established by right middle cerebral artery occlusion for 1 h and reperfusion for 24 h. EA (20 Hz/80 Hz, 1-3 mA) was applied to "Baihui" (GV 20) and "Dazhui" (GV 14) for 30 min. The ischemic cerebral cortex tissue was taken, fixed in 2.5% glutaral solution, embedded in araldite, cut into sections and stained for observing the ultrastructure changes of cortical pyramidal cells, astrocyte and blood brain barrier (BBB) under transmission electronic microscope. RESULTS: In sham group, the anatomical structure of the cerebral pyramidal cells was normal basically. In model group, the neuronal mitochondria and the capillary endothelium and the processes of the astrocyte got swelling, the mitochondrial cristae were broken, the capillary lumens became narrow or were blocked up. In EA group, the injury degree of the pyramidal cells, glial cells and BBB were lighter. CONCLUSION: EA can reduce ischemic injury of the cerebral cortical neurons and blood brain barrier in rats with CI/R injury.

[Involvement of melatonin in the adjusting effect of electroacupuncture in resisting oxygen stress in cerebral ischemia-reperfusion injury rats].

OBJECTIVE: To explore the effect of electroacupuncture (EA) in resisting acute cerebral ischemia-reperfusion injury (CI-RI) via anti-oxidation of melatonin (MT). METHODS: A total of 52 male SD rats were randomized into Sham-operation (sham, n=8), CI-RI (model, n=8), EA (n=8), MT (3.2 mg/Kg, i.p., n=8), EA+MT (n=8), EA+Luzindole (L, a blocker of MT, 1 mg/100 microl/rat, n=6), EA+PD 98059 (a blocker of mitogen-activated protein kinase, MAPK, 1 mg 100 microl/rat, n=6) groups. EA (3 Hz,1-3 mA, continuous waves) was applied to "Dazhui" (GV 14) and "Baihui" (GV 20) for 30 min. CI-RI model was induced by right middle cerebral artery occlusion (MCAO) for 60 min and reperfusion for 24 h except sham-operation group. MT content of pineal gland tissue was determined by high performance liquid chromatography (HPLC) and neuroethological scores of the rats were evaluated by Kuluz's and Julio's methods. Bax and Bcl-2 gene protein expression of the striate body was detected by immunohistochemistry. RESULTS: In comparison with sham group, the number of Bax immunoreactive (IR) positive cells and Bax/Bcl-2 in striate body increased significantly (P<0.001), and neuroethological score and Bcl-2 IR-positive cells decreased considerably in model group (P<0.01). Compared with model group, pineal MT content, neuroethological score and Bcl-2 IR-positive cells in EA group, neuroethological scores in EA+L and EA+P groups, and Bcl-2 expression in MT and EA+MT groups increased considerably (P<0.01, 0.001); while Bax IR-positive cells and Bax/Bcl-2 in EA, MT and EA+MT groups decreased very significantly (P<0.001). It showed that EA could obviously increase pineal MT content, improve CI-RI rats' activity score, upregulate Bcl-2 expression and down-regulate Bax expression in striate body. In comparison with simple EA group, neuroethological scores of EA+L and EA+P groups, Bax IR-positive cells and Bax/Bcl-2 of EA+MT group were significant lower (P<0.05, 0.01). No significant differences were found between EA+L and EA+P groups in behavior scores, and between MT and EA+MT groups in Bax and Bcl-2 IR-positive cell number and Bax/Bcl-2 (P>0.05). CONCLUSION: EA has a good effect of anti-oxygen stress, protecting the brain from ischemic damage directly or indirectly, which may be related to its effect in upregulating the synthesis and release of MT.

Progress in researches on the effect of acupuncture in antagonizing oxygen stress.

Oxidation and free radicals participate in the pathological process of multiple diseases in organisms, and acupuncture shows good effect in antagonizing oxygen stress (OS). This article reviews the effect of acupuncture in antagonizing oxygen stress and the mechanism of its anti-free radical effect in various diseases. The authors hold that acupuncture not only has a chain-blocking effect, but also has preventive and repairing effects of anti-oxidation. And anti-OS action is one of the important mechanisms of acupuncture.