Effect of "Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion on the expression of Fas/FADD/Caspase-8 of death receptor pathway in rats with primary ovarian insufficiency / 中国针灸

OBJECTIVE@#To explore the effect of "Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion on the ovarian function in the rats with primary ovarian insufficiency (POI) and the potential effect mechanism based on the Fas/FADD/Caspase-8 of death receptor pathway.@*METHODS@#Forty-eight female SD rats were randomly divided into a blank group, a model group, a medication group and an acupuncture group, with 12 rats in each group. Except in the blank group, the rats in the other groups were intraperitoneally injected with cyclophosphamide to establish the POI model. In the acupuncture group, after successful modeling, the intervention was given with "Zhibian" (BL 54)-to- "Shuidao" (ST 28) needle insertion, once daily, 30 min in each intervention; and the duration of intervention was 4 weeks. In the medication group, estradiol valerate tablets were administered intragastrically, 0.09 mg•kg-1•d-1, for 4 weeks. The general situation and the estrous cycle of the rats were compared among groups. Using ELISA, the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) in the serum were detected. HE staining was adopted to observe the morphological changes of ovarian tissue of rats. The protein expression of Fas, FADD and Caspase-8 in ovarian tissue was detected with immunohistochemistry and Western blot.@*RESULTS@#After modeling, except the rats of the blank group, the rats of the other groups had dry fur, lost hair, low spirits, reduced food intake, increased urination and loose stool. After intervention, the stool became regular gradually in the acupuncture group and the medication group. The percentage of estrous cycle disturbance was increased in the rats of the model group when compared with the blank group (P<0.01); in comparison with the model group, the percentages of estrous cycle disturbance were reduced in the acupuncture group and the medication group after intervention (P<0.01). When compared with the blank group, the body mass and E2 content in the serum were lower (P<0.01), the levels of FSH and LH in the serum and the protein expression levels of Fas, FADD and Caspase-8 were increased (P<0.01) in the model group. Compared with the model group, the body mass and E2 contents in the serum were higher (P<0.01), the levels of FSH and LH in the serum and the protein expression levels of Fas, FADD and Caspase-8 were reduced (P<0.01) in the acupuncture group and the medication group.@*CONCLUSION@#"Zhibian" (BL 54)-to-"Shuidao" (ST 28) needle insertion can effectively improve the ovarian function of POI rats, and its effect mechanism may be related to regulating the serum sex hormone levels, reducing the expression of Fas, FADD and Caspase-8 in ovarian tissue and retarding apoptosis of ovarian cells.

Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on the expression of apoptosis-related factors in rats with premature ovarian insufficiency based on oxidative stress / 中国针灸

OBJECTIVE@#To explore the possible mechanism of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on premature ovarian insufficiency (POI) from the perspective of oxidative stress.@*METHODS@#Sixty female SD rats were randomly divided into a blank group, a model group, a sham acupuncture group, a medication group, and an acupuncture group, 12 rats in each group. Except the blank group, the rats in the remaining groups were intraperitoneally injected with cyclophosphamide to establish the POI model. After the model was successfully established, the rats in the acupuncture group were treated with acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28), with a depth of about 12 mm, and the needle was retained for 30 min; the acupuncture was given once a day, for a total of 4 weeks. The rats in the sham acupuncture group were treated with blunt-head needle to tap the skin surface of "Zhibian" (BL 54), without penetrating the skin, once a day for 4 weeks. The rats in the medication group were treated with estradiol valerate by gastric gavage for 4 weeks. After the intervention, the level of reactive oxygen species (ROS) in the ovarian tissue was detected by fluorescence probe; the expression of c-Jun N-terminal kinase (JNK), forkhead box O1 (FoxO1), tumor suppressor gene protein 53 (p53) and p53 up-regulated modulator of apoptosis (Puma) mRNA and protein in ovarian tissue were detected by real-time fluorescence quantitative PCR and Western blot.@*RESULTS@#Compared with the blank group, the level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the model group were increased (P<0.01). Compared with the model group, the level of ROS and the expression of p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the sham acupuncture group were slightly reduced, but the difference was not statistically significant (P>0.05). The level of ROS and the expression of JNK mRNA, p-JNK protein, FoxO1, p53, Puma mRNA and protein in the ovarian tissue in the acupuncture group and the medication group were reduced (P<0.01).@*CONCLUSION@#Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could improve the level of oxidative stress, down-regulate the expression of apoptosis-related factors JNK, FoxO1, p53 and Puma induced by oxidative stress, and inhibit the premature failure of ovarian reserve function caused by apoptosis of ovarian granulosa cells in POI rats.

Effect of Buyang Huanwu Decoction on cerebral ischemia-reperfusion injury in rats by regulating autophagy through PI3K/AKT pathway / 中国药理学通报

Aim To explore the effect of Buyang Huanwu Decoction on cerebral ischemia-reperfusion injury in rats by regulating autophagy through PI3K/AKT pathway. Methods The rats were randomly divided into five groups(n=10): sham operation group(Sham), model group(Model), Buyang Huanwu Decoction group(BYHWD), PI3K inhibitor group(LY294002)and Vehicle group(Vehicle). Except Sham group, the other groups were treated with 2h ischemia and 72 h reperfusion for modeling. The Zea Longa score was used to assess the neurological defects, HE was used to observe brain injury in the ischemic penumbra(IP), immunofluorescence was employed to detect LC3, and Western blot was used to detect pathway and autophagy marker proteins. Results Compared BYHWD group with model group, the neurological score of rats decreased, cerebral infarction volume decreased, the pathological lesions of brain IP were relieved, PI3K and p-AKT/AKT expression increased, and LC3Ⅱ/ decreased and p62 increased(P<0.05). The regulatory effect of BYHWD was weakened by LY294002(P<0.05). Conclusion Buyang Huanwu Decoction alleviates cerebral ischemia-reperfusion injury in rats by activating PI3K/AKT pathway to inhibit autophagy.

Mechanism of Buyang Huanwu Decoction alleviating inflammatory response after cerebral ischemia/reperfusion injury by activating SIRT1 in hippocampus of rats / 中国药理学通报

Aim To explore the inhibitory effect of Buyang Huanwu Decoction on the inflammatory response in the hippocampus of brain tissues of CIRI rats by regulating SIRT1 and the underlying mechanism. Methods The middle cerebral artery embolization (MCAO) model was prepared in rats and divided into sham operation group (Sham), model group (MCAO/R), Buyang Huanwu Decoction group (BYHWT),and BYHWT + SIRT1 inhibitor group (BYHWT + EX527). Zea Longa was used to detect the neurological function score of rats in each group; TTC staining was used to determine the volume of cerebral infarction; HE staining was used to observe the pathological damage of the hippocampus; Western blot was used to detect the expression levels of SIRT1 and IL-6; immunohistochemistry was used to detect TNF-α, IL-1β expression level. Results Compared with the sham group,the neurological function score of the MCAO/R group increased (P < 0.05); the volume of cerebral infarction increased (P < 0.05); the nerve cells in hippocampus were severely damaged, arranged disorderly, and the nucleus was broken; Western blot showed that the expression of SIRT1 decreased, IL-6 expression increased (P <0.05); immunohistochemistry showed that TNF-α,IL-1β expression increased (P < 0.05). Compared with the MCAO/R group, the neurological function score of the BYHWT group decreased (P <0.05); the volume of cerebral infarction decreased (P < 0.05); the damage of nerve cells in hippocampus was reduced; Western blot showed that the expression of SIRT1 increased and IL-6 expression decreased (P < 0.05); immunohistochemistry showed that TNF-α, IL-1β expression decreased (P < 0.05). Compared with the BYHWT group, the neurological function score of the BYHWT + EX527 group increased (P < 0.05); the volume of cerebral infarction was raised (P <0.05); the damage of nerve cells in hippocampus was aggravated; Western blot showed that the expression of SIRT1 decreased and IL-6 expression increased (P < 0.05); immunohistochemistry showed that TNF-α, IL-1β expression increased (P < 0.05). Conclusions Preliminary discussion of Buyang Huanwu Decoction can activate SIRT1 in hippocampus of rat brain tissues to reduce the inflammatory response after CIRI and play a role in brain protection.

Effect of Buyang Huanwu decoction on expression of silent information regulator 1 in cortical area of rats after cerebral ischemia/reperfusion injury / 中国药理学通报

Aim To investigate the effect of Buyang Huanwu decoction on the expression of silencing regulation factor 1(SIRT1)protein in cortical area and the possible mechanism of cerebral ischemia/reperfusion injury(CIRI)via establishing middle cerebral artery occlusion(MCAO)model. Methods SD rats were randomly divided into sham group(Sham), model group(MCAO/R), Buyang huanwu decoction group(BYHWT), and atorvastatin group(Atorvastatin), with 15 rats in each group. After 2 h ischemia/reperfusion for 72 h and drug intervention, the model was successfully constructed by using laser speckle blood flow monitoring video system. Zea Longa neurological function score was used to evaluate the neurological defects of rats after modeling. TTC staining was used to detect infarct volume. Nissl staining was used to observe the injury of nerve cells. Western blot was employed to detect the SIRT1 protein expression level. Immunofluorescence was applied to detect the fluorescence expression of SIRT1. Results Compared with sham group, the neurological deficits of MCAO/R group were serious(P<0.05). Cerebral infarction volume increased(P<0.05). The nerve cells were severely damaged, disordered, with the nucleus pyknosis(P<0.05). SIRT1 protein expression was reduced(P<0.05). The fluorescence intensity of SIRT1 decreased(P<0.05). Compared with MCAO/R group, the neurological impairment degree of rats in BYHWT and Atorvastatin groups was reduced(P<0.05). The proportion of cerebral infarction volume decreased(P<0.05). The injury of nerve cells was significantly reduced and the number of nerve cells increased(P<0.05). The expression of SIRT1 protein was up-regulated(P<0.05). Fluorescence intensity of SIRT1 increased(P<0.05). Conclusions Buyang huanwu decoction can effectively alleviate brain injury in cerebral ischemia/reperfusion rats, and its protective effect may be related to the increase of SIRT1 protein expression in the ischemic cortical region.

Buyang huanwu decoction attenuates cerebral ischemia-reperfusion injury in rats by regulating autophagy through AMPK/mTOR/ULKl signaling pathway / 中国药理学通报

Aim To explore the mechanism of Buyang huanwu decoction attenuating cerebral ischemia-reper- fusion injury in rats by regulating autophagv through AMPK/mTOR/ULKl signaling pathway.Methods Left eerebral ischemia model in rats was established by modified thread ocelusion method, then the rats in each group were given medieine onee every 24 hours for 3 times.After 72 hours of reperfusion, the nerve injury and the changes of cerebral infarction volume were observed; the morphology, number and apoptosis of nerve cells were observed by Nissl staining and TUNEL staining; the expression of autophagy protein and AMPK/mTOR/LJLKl autophagy signaling pathway related proteins were detected by Western blot.Results Buyang huanwu decoction could improve the neurological deficit of rats, reduce the volume of cere bral infarction and neuronal apoptosis, reduce the pathological injury of brain tissues, inhibit the phosphorylation activation of AMPK, relieve the inhibition of AMPK on downstream mTOR and LJLK1 , promote the phosphorylation activation of both, and inhibit autophagy.AMPK agonist metformin increased the level of autophagy and reversed the protective effect of Buyang huanwu decoction on cerebral ischemia-reperfusion injury in rats.Conclusion Buyang huanwu decoction mediates AMPK/mTOR/ULKl autophagy signaling pathway to play a neuroprotective effect on cerebral is- chemia-reperfusion injury in rats.

Effect of calycosin in reducing cerebral ischemia/reperfusion injury in rats by regulating cytochrome C/apoptotic protease activating factor-1 apoptosis signal pathway / 解剖学报

Objective To investigate the effect of calycosin on cerebral ischemia/reperfusion injury and its mechanism. Methods Forty SPF male SD rats were randomly divided into sham group, model group, calycosin group (20 mg/kg), nimodipine group (0.7 mg/kg, positive control group). The occlusion model of middle cerebral artery in rats was established by modified thread occlusion method, and the environment of cerebral ischemia-reperfusion injury was simulated in vivo. Zea longa score was used to detect the neurological deficit of rats after ischemia-reperfusion injury, 2, 3, 5-triphenyltetranitrogen (TTC) was used to detect the volume of cerebral infarction, HE staining was used to detect the pathomorphological changes of nerve cells, Nissl staining was used to observe the changes of nissl bodies, TUNEL staining was used to detect the apoptosis of nerve cells, Western blotting was used to detect the expression of cytochrome C (Cyt C), apoptotic protease activating factor-1 (Apaf-1), Caspase-9 and Caspase-3. Results Compared with the sham group, the neurological deficit symptoms in the model group were significant (P<0.05), the volume of cerebral infarction increased significantly (P<0.05). Under the microscope, it was found that the nerve cells showed contraction of cell body, hyperchromatic and pyknosis of nucleus and poor growth state, the expression of nissl body reduced significantly (P < 0.05), the apoptotic nerve increased significantly (P< 0.05), the expression of Cyt C, Apaf-1, Caspase-9 and Caspase-3 increased significantly (P<0.05). Compared with the model group, the neurological deficit symptoms of calycosin group and nimodipine group reduced significantly (P<0.05), the volume of cerebral infarction reduced significantly (P<0.05). Under the microscope, the damage of nerve cells reduced significantly, the expression of nissl body increased significantly (P<0.05), the apoptotic nerve reduced significantly (P<0.05), the expression of Cyt C, Apaf-1, Caspase-9 and Caspase-3 decreased significantly (P<0.05). Conclusion Calycosin can significantly inhibit the apoptosis of nerve cells and reduce the cerebral ischemia-reperfusion injury. Its mechanism of action is related to the effective regulation of Cyt C/Apaf-1 apoptosis signaling pathway by calycosin.

Herb-separated moxibustion on dysmenorrhea in ovarian endometriosis: a randomized controlled trial / 中国针灸

OBJECTIVE@#To observe the clinical therapeutic effect of herb-separated moxibustion on dysmenorrhea in ovarian endometriosis.@*METHODS@#A total of 54 patients with ovarian endometriosis dysmenorrhea were randomized into a herb-separated moxibustion group and a waiting-list group, 27 cases in each one (3 cases dropped off in the herb-separated moxibustion group, 4 cases dropped off in the waiting-list group). Herb-separated moxibustion was applied at hypogastrium and lumbosacral area for 30 min in the herb-separated moxibustion group, once a week for 3 months, and oral ibuprofen sustained-release capsule was given to relieve pain when necessary. Excepting giving ibuprofen sustained-release capsule when necessary, no more intervention was adopted in the waiting-list group. Before and after treatment and in 3 months follow-up, visual analogue scale (VAS) score, days of dysmenorrhea, total dose of oral painkiller were observed.@*RESULTS@#Compared before treatment, the VAS scores after tratment and in follow-up were decreased in the herb-separated moxibustion group (<0.05), and were less than those in the waiting-list group (<0.05); the days of dysmenorrhea and the total doses of oral painkiller after tratment and in follow-up were decreased in the herb-separated moxibustion group (<0.05), and were less than those in the waiting-list group (<0.05).@*CONCLUSION@#Herb-separated moxibustion can effectively improve dysmenorrhea symptom and shorten dysmenorrhea days in patients with ovarian endometriosis.

Effects of calycosin on mitochondrial apoptotic pathway in oxygen-glucose deprivation / reoxygenation PC12 cells / 解剖学报

Objective To investigate the effect of calycosin on mitochondrial apoptotic pathway in oxygen-glucose deprivation/ reoxygenation PC12 cells. Methods PC12 cells were randomly divided into four groups: control group, model group, calycosin group and nimodipine group. Except for the control group, the other groups were treated with oxygen and glucose deprivation for 2 hours and compound oxygen and glucose for 24 hours. Calycosin group and nimodipine group were treated with drug-containing medium containing calycosin (0. 07 μmol/ L) and nimodipine (5. 00 μmol/ L) simultaneously with reoxygenation. CCK-8 method was used to detect cell survival rate, flow cytometry was used to detect cell apoptosis rate, immunofluorescence method was used to detect Bax/ Bcl-2 ratio, Western blotting was used to detect the expression of key proteins cytochrome C (Cyt-C), apoptotic protease activating factor-1 (Apaf-1) and Caspase-3 in mitochondrial apoptotic pathway. Results Compared with the control group, the survival rate of cells in model group decreased significantly (P<0. 05), and the apoptotic rate increased significantly (P<0. 05), the ratio of Bax/ Bcl-2 was significantly increased (P<0. 05), and the expression of key proteins Cyt-C, Apaf-1 and Caspase-3 in mitochondrial apoptotic pathway were significantly increased (P<0. 05). Compared with the model group, the cell survival rates of calycosin group and nimodipine group increased significantly (P < 0. 05), apoptotic rate decreased significantly (P<0. 05), the ratio of Bax/ Bcl-2 was significantly decreased (P<0. 05), and the expression of key proteins of mitochondrial apoptotic pathway, Cyt-C, Apaf-1 and Caspase-3 were significantly decreased (P < 0. 05). The difference has statistical significance. Conclusion Calycosin can significantly improve the survival rate of oxygen-glucose deprivation/ reoxygenation PC12 cells and inhibit cell apoptosis. Its mechanism is closely related to the inhibition of the expression of key proteins Cyt-C, Apaf-1 and Caspase-3 in mitochondrial apoptotic pathway by calycosin.

Study of astragaloside IV alleviating oxidatIVe stress injury of pc12 cells induced by oxygen-glucose deprIVation/reoxygenation through regulating autophagy / 中国药理学通报

Aim To observe the effects of astragaloside IV on autophagy and oxidative stress induced by oxy gen-glucose deprivation/reoxygenation in PCI2 cells. Methods PCI2 cells were divided into normal group, model group (ox-ygen-glucose deprivation/reoxygenation group) , astragaloside IV group, and autophagy inhibitor + astragaloside IV group. CCK-8 was used to detect cell viability, and ELISA to detect MDA, SOD and GSH-Px. Transmission electron microscope and MDC fluorescent staining were employed to observe the changes of au-tophagosome, and Western blot to detect the protein expression of Beclinl. Results Compared with normal group, the cell activity in model group decreased (P <0.05), MDA content increased, SOD and CSH-Px activity decreased (P < 0.05), and autophagosomes could be seen and the protein expression of Beclinl increased ( P < 0.05). Compared with model group, the cell activity in astragaloside IV group increased ( P < 0. 05), MDA content decreased, SOD and GSH-Px activity increased (P < 0.05), the number of autophagosomes and the protein expression of Beclinl increased (P<0.05). When autophagy inhibitor was given at the same time, the autophagy inhibitor could obviously antagonize the antioxidant effect of astragaloside IV while alleviating autophagy. Conclusions Astragaloside IV can protect PC 12 cells from oxidative stress injury induced by oxygen-glucose deprivation/reoxygenation by up-regulating autophagy.

Comparative study on extraction and culture method of neural stem cells / 中国药理学通报

Aim To explore the best method of neural stem cellextraction and culture, and provide a technical reference for thebasic research of neural stem cells. Methods Different extractionand culture methods of neural stem cells were compared.The rate of ball of neural stem cells and the stability of neuralstem cells in undifferentiated state were observed by extraction offetal and neonatal rats cortex, using different types of medium,different inoculation density, different culture methods, differentmethods of changing liquid and different passage methods. Neuralstem cells' activities were detected by Varioskan LUX MultimodeMicroplate Reader. Results ① The brain cortex of fetalrats of 14 d had higher proportion of neural stem cells, less othercells and more neurospheres than newborn rats of 24 h. ② Neuralstem cells could be stabilized in undifferentiated state by usingserum-free medium, while most of the neural stem cellswere differentiated into neurons and glial cells by using serummedium. ③ Neural stem cells, seeding at 1. 0 ×109 ·L-1 , hada large number of neurosperes and were in good condition. ④Suspension culture was beneficial to form a stable neurosphereand keep the neural stem cells in an undifferentiated state thanadherent culture. ⑤ The state of neurosphere by changing halfof the medium and adding medium without discarding was betterthan that by replacing all medium. ⑥ The neurospheres couldbe separated into single cells by mechanical blow in primary generationand the second generation of neurospheres cultured in serum-free medium. While the percentage of viable cells in neuralstem cells was the highest digested with stem cell lysates afterthree generations and the neurospheres re-formed were better. ⑦Neural stem cells' activity of 14 d fetal rat in Accutase digestiongroup was significantly higher than that of the other threegroups, and the difference was significant (P <0. 05). ConclusionsNeural stem cells proliferate and divide well, with highrate of ball formation and good neurosphere condition, which canmaintain a stable undifferentiated state by extracting the cerebralcortex of 14 d fetal rats, using serum-free medium, inoculatinginto a 25 cm2 flask at a density of 1. 0 × 109 ·L-1 , and takingthe suspension culture (adding the medium 1 ~ 1. 5 mL every 2~3 d and passage every 6 ~8 d ).

Effects of electroacupuncture at points "Weibingfang" on EGF and EGFR mRNA expressions in rats with lesion of gastric mucosa / 中国针灸

<p><b>OBJECTIVE</b>To probe the mechanism of electroacupuncture at points "Weibingfang" in treatment of acute gastric mucosal lesion.</p><p><b>METHODS</b>Forty Wistar rats of sanitary degree were randomly divided into 4 groups, normal group, model group, sham-model group and Weibingfang group, 10 rats in each group. The acute gastric mucosal lesion model was made by intragastric perfusion of anhydrous alcohol. The Weibingfang group were treated by electroacupuncture at "Neiguan" (PC 6), "Zhongwan" (CV 12) and "Zusanli" (ST 36) with sparse-dense wave, frequency of 10-30 Hz, current intensity of 2 mA, for 20 min. One hour after the treatment, the blood from the abdominal aorta and the gastric mucosa tissue were taken, and serum epidermal growth factor (EGF) level and epidermic growth factor receptor mRNA (EGFR mRNA) expression in the gastric mucosa were detected with enzyme linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively.</p><p><b>RESULTS</b>There were significant differences in blood EGF level (41.62 +/- 12.58) ng/L and EGFR mRNA expression (0.78 +/- 0.03) in the model group were significantly different from [(60.37 +/- 12.01) ng/L and 0.55 +/- 0.04] in the normal group and [(61.21 +/- 13.46) ng/L and 0.53 +/- 0.05] in the sham-model group (P < 0.05); after electroacupuncture, blood EGF level (70.59 +/- 10.14) ng/L increased and the EGFR mRNA expression (1.18 +/- 0.02) in the gastric mucosa was up-regulated with a significant differences as compared with those in the model group (P < 0.05).</p><p><b>CONCLUSION</b>Point "Weibingfang" can promote proliferation, differentiation and migration of the gastric mucosal epidermic cells to repair the gastric mucosal lesion.</p>