[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].

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].

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 penetrative needling of "Zhibian" (BL54) through "Shuidao" (ST28) on expression of TRAIL and its receptors in rats with premature ovarian insufficiency].

OBJECTIVE: To observe the effect of penetrative needling of "Zhibian" (BL54) through "Shuidao" (ST28) on the expressions of death receptor pathway-related protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, as death receptor 4 (DR4), death receptor 5 (DR5), decoy receptor 1 (DcR1) and decoy receptor 2 (DcR2) in premature ovarian insufficiency (POI) rats, so as to explore its mechanisms underlying improvement of POI. METHODS: Forty female SD rats were randomly divided into blank control, model, penetrative needling and medication (estradiol valerate) groups, with 10 rats in each group. The POI model was established by intraperitoneal injection of cyclophosphamide (D1: 50 mg·kg-1·d-1, D2 to D15: 8 mg·kg-1·d-1, for a total of 15 d). After successful modeling, the rats in the penetrative needling group received penetrative needling of BL54 through ST28, with the needle retained for 30 min, once a day for a total of 4 weeks. Rats of the medication group received gavage of estradiol valerate (0.09 mg·kg-1·d-1) once daily for 4 weeks. After the intervention, the content of serum follicles of stimulation hormone (FSH),lateinizing hormone (LH),estradiol (E2) and vascular endothelial growth factor (VEGF) were assayed using enzyme-linked immunosorbent assay, and histopathological changes of ovarian tissue and the number of follicles were observed under light microscope after H.E. staining. The expression levels of TRAIL, DR4, DR5, DcR1, DcR2, and Fas-associated death domain (FADD) in ovarian tissues were detected using quantitative real-time PCR, and the immunoactivity of ovarian TRAIL, DR4 and DR5 detected using immunohistochemistry. The body weight and the damp weight of ovary were measured for calculating the ovarian coefficient. RESULTS: Compared with the blank control group, the E2 and VEGF contents, ovarian coefficient, and the number of the primary, secondary and sinus follicles were significantly decreased (P<0.01) in the model group, whereas FSH and LH contents, the atretic follicle number, TRAIL, DR4 and DR5 immunoactivity, and the expression levels of TRAIL, DR4, DR5 and FADD mRNAs considerably increased in the model group (P<0.01). In comparison with the model group, the decrease of the VEGF content, ovarian coefficient, and the number of the primary, secondary and sinus follicles, and the increase of the atretic follicle number, TRAIL, DR4 and DR5 immunoactivity, and expression levels of TRAIL, DR4, DR5 and FADD mRNAs were reversed in both penetrative needling and medication groups (P<0.01, P<0.05). The number of primary follicles was significantly more in the medication group than in the penetrative needling group (P<0.01). CONCLUSION: Penetrative needling of BL54 and ST28 can improve ovarian weight and promote follicular development in POI rats, which may be associated with its function in down-regulating the expression of pro-apoptotic proteins TRAIL, DR4, DR5 and FADD of the death receptor pathway to inhibit apoptosis of ovarian granulosa cells.

[Herb-separated moxibustion on dysmenorrhea in ovarian endometriosis: a randomized controlled trial].

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 (P<0.05), and were less than those in the waiting-list group (P<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 (P<0.05), and were less than those in the waiting-list group (P<0.05). CONCLUSION: Herb-separated moxibustion can effectively improve dysmenorrhea symptom and shorten dysmenorrhea days in patients with ovarian endometriosis.

The Role of Astragaloside IV against Cerebral Ischemia/Reperfusion Injury: Suppression of Apoptosis via Promotion of P62-LC3-Autophagy.

Background: Ischemia/reperfusion (I/R) caused by ischemic stroke treatments leads to brain injury, and autophagy plays a role in the pathology. Astragaloside IV is a potential neuroprotectant, but its underlying mechanism on cerebral I/R injury needs to be explored. The objective of this study is to investigate the neuroprotective mechanism of Astragaloside IV against cerebral I/R injury. Methods: Middle cerebral artery occlusion method (MCAO) and oxygen and glucose deprivation/reoxygenation (OGD/R) method were used to simulate cerebral I/R injury in Sprague-Dawley (SD) rats and HT22 cells, respectively. The neurological score, 2,3,5-Triphe-nyltetrazolium chloride (TTC) staining, and transmission electron microscope were used to detect cerebral damage in SD rats. Cell viability and cytotoxicity assay were tested in vitro. Fluorescent staining and flow cytometry were applied to detect the level of apoptosis. Western blotting was conducted to examine the expression of proteins associated with autophagy. Results: This study found that Astragaloside IV could decrease the neurological score, reduce the infarct volume in the brain, and alleviate cerebral I/R injury in MCAO rats. Astragaloside IV promoted cell viability and balanced Bcl-2 and Bax expression in vitro, reduced the rate of apoptosis, decreased the expression of P62, and increased the expression of LC3II/LC3I in HT22 cells after OGD/R. Conclusions: These data suggested that Astragaloside IV plays a neuroprotective role by down-regulating apoptosis by promoting the degree of autophagy.

Effects of rehabilitation training on apoptosis of nerve cells and the recovery of neural and motor functions in rats with ischemic stroke through the PI3K/Akt and Nrf2/ARE signaling pathways.

This study was designed in order to investigate the effects between rehabilitation training on the apoptosis of nerve cells and the recovery of neural and motor functions of rats with ischemic stroke by way of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and nuclear factor E2-related factor 2/antioxidant responsive element (Nrf2/ARE) signaling pathways. In total, 110 healthy adult male Sprague-Dawley (SD) rats were selected in order to take part in this study. Ninety SD rats were used in order to establish the middle cerebral artery occlusion (MCAO), among which 80 rats were randomly assigned as part of the natural recovery, natural recovery+Rp-PI3K (the rats injected with PI3K/Akt inhibitor LY294002), rehabilitation training, and rehabilitation training+Rp-PI3K groups. Meanwhile, 20 rats were selected as part of the sham operation group. The neural and motor functions of these rats were evaluated using a balance beam test and the Bederson score. The mRNA expressions of PI3K, Akt, Nrf2 and HO-1 were measured using an RT-qPCR. The protein expressions of PI3K, p-PI3K, Akt, p-Akt, Nrf2 and HO-1 were also detected by using western blotting and the immunohistochemistry process. The cell cycle and cell apoptosis were detected by using a flow cytometry and TUNEL assay. The sham operation group exhibited lower neural and motor function scores than other groups. At the 7, 14, and 21 d marks of this study, the neural and motor function scores were increased in the natural recovery, natural recovery+Rp-PI3K, and rehabilitation training+Rp-PI3K groups in comparison with the rehabilitation training group but found to be decreased in the natural recovery group in comparison with the natural recovery+Rp-PI3K group. In comparison with the sham operation group, expressions of PI3K, Nrf2 and HO-1, and proportions of p-PI3K/PI3K and p-Akt/Akt were all higher in the natural recovery, rehabilitation training, and rehabilitation training+Rp-PI3K groups. Same trends were found in the rehabilitation training group in comparison with the natural recovery and rehabilitation training+Rp-PI3K groups, as well as in the natural recovery group in comparison with the natural recovery+Rp-PI3K group. In comparison with the sham operation and rehabilitation training groups, hippocampal nerve cells at G1 phase and the cells apoptosis were both elevated in the other three groups which were found to be decreased in the natural recovery group in comparison with the natural recovery+Rp-PI3K group. Our results indicated that the rehabilitation training can inhibit the apoptosis of nerve cells as well as promote the recovery of both neural Rehabilitation training in rats with IS.

[Effects of electroacupuncture at acupoints "changbingfang" on serum IL-4 content and colonic NF-kappaB p65 expression in rats with ulcerative colitis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zhongwan"(CV 12), "Tianshu"(ST 20) and "Shangjuxu"(ST 37) (an acupoint prescription "Changbingfang" for treatment of intestinal disorders) on the expression of colonic nuclear factor kappaB p65 (NF-kappaB p65) protein expression and serum interleukin-4 (IL-4) content in rats, so as to explore its mechanism underlying improvement of ulcerative colitis (UC). METHODS: Thirty SD rats were randomly and equally divided into normal control, model and EA groups (10 rats/group). The UC model was made by enema with 8% acetic acid. EA stimulation (6 Hz/30 Hz) was delivered to "Zhongwan" (CV 12), "Tianshu" (ST 20) and "Shangjuxu" (ST 37) for 15 min separately, 0 h, 24 h and 48 h post-enema. The blood and colon tissues were taken for detection of serum IL-4 content with ELISA, and for assaying colonic NF-kappaB p65 protein expression with immunohistochemistry and Western blot, respectively. RESULTS: Compared with the normal control group, colonic NF-kappaB p65 protein expression level shown by immunohistochemistry and Western blot and serum IL-4 content were significantly higher in the model group (P < 0.01, P < 0.05). After EA stimulation, colonic NF-kappaB p65 protein expression levels shown by both immunohistochemistry and Western blot were down-regulated significantly (P < 0.01, P < 0.05) and serum IL-4 content was up-regulated further in comparison with the model group (P < 0.01). CONCLUSION: EA of acupoint recipe "Changbingfang" can down-regulate the proinflammatory NF-kappaB p65 protein expression and up-regulate serum anti-inflammatory cytokine IL-4 level in rats, which may contribute to its effect in improving ulcerative colitis.

[Effect of heating time on the chemical composition of bamboo juice by GC-MS].

OBJECTIVE: To study the effect of heating time on the chemical composition of bamboo juice. METHODS: GC-MS was used to analyze the chemical components of bamboo juice collected from the bamboo heating for 10 min and 50 min. RESULTS: 153 peaks were detected from the bamboo juice of 10 min. Among of them, 36 peaks were identified. As for the bamboo juice of 50 min, 80 peaks were detected and 29 identified. CONCLUSION: Heating time has effect on the chemical composition of bamboo juice, the substances detected in short time are more than that in long time.

[Selection of basic acupoints for composing "gastric-disorder-formula" for electroacupuncture prevention of acute gastric mucosal lesion in rats].

OBJECTIVE: To select the best combination of the commonly-used acupoints [Zhongwan (CV 12), Neiguan (PC 6), and Zusanli (ST 36)] for treatment of gastric disorders. METHODS: Forty-eight Wistar rats were randomly divided into 8 groups: model, ST36, CV12, PC6, ST36 + PC6, ST36 + CV12, CV12 + PC6 and ST36 + CV12 + PC6 by factorial design method. Acute gastric mucosal lesion model was established by intragastric perfusion of anhydrous alcohol. EA(1- 3 mA, 30 Hz) was applied to the above-mentioned acupoints for 20 min respectively. And after EA, gastric mucosal lesion index (LI), pathological lesion integral score (PLIS) were calculated and mucosal ultramicrostructural changes observed by optical microscope and transmission electron microscope respectively. RESULTS: Compared with model group, the gastric mucosal LI and PLIS of ST36, CV12, PC6, ST36 + CV12, ST36 + PC6, CV12 + PC6, ST36 + CV12 + PC6 groups all decreased significantly (P < 0.05). Comparison among 7 EA groups showed that both LI and PLIS of ST36 + CV12 + PC6 group were significantly lower than those of ST36, V12, PC6, ST36 + CV12, ST36 + PC6 and CV12 + PC6 groups (P < 0.05). No significant differences were found between every two groups of ST36, CV12, PC6, ST36+ CV12, ST36 + PC6 and CV12 + PC6 groups (P > 0.05) in both LI and PLIS. Ultrastructural results showed that the injury of gastric mucosal parietal cells and delomorphous cells including mitochondrial swelling, crista arrange disorder and breaking, enlargement of the rough endoplasmic reticulum, ribosome exfoliation, etc. in model group was serious, but lightened significantly in EA groups particularly in ST36 + CV12 + PC6 group. CONCLUSION: ST36, PC6 and CV12 combination can effectively reduce anhydrous alcohol-induced gastric mucosal lesion in rats, and may be used as a basic formula

[Effects of electroacupuncture at points "Weibingfang" on EGF and EGFR mRNA expressions in rats with lesion of gastric mucosa].

OBJECTIVE: To probe the mechanism of electroacupuncture at points "Weibingfang" in treatment of acute gastric mucosal lesion. METHODS: 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. RESULTS: 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). CONCLUSION: Point "Weibingfang" can promote proliferation, differentiation and migration of the gastric mucosal epidermic cells to repair the gastric mucosal lesion.