[Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) combined with Zhuang-medicine-thread moxibustion on expression of apoptosis-related factors in gastric antrum of diabetic gastroparesis (DGP) rats, so as to explore its mechanism underlying improvement of DGP. METHODS: Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion (moxibustion) and EA+moxibustion (combination) groups (12 rats in each group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension （10 mL/kg）. EA (10 Hz/50 Hz, 2 mA, 20 min) or Zhuang-medicine-thread moxibustion (3 cones) was applied to "Zhongwan" (CV12), bilateral "Neiguan" (PC6) and bilateral "Sanyinjiao" (SP6) of the related groups, once a day for 3 weeks. The blood glucose, gastric emptying rate and intestinal propulsion rate of rats were measured. The apoptosis index of gastric antrum cells were observed by TUNEL staining. The protein and mRNA expressions of Caspase-3, B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in gastric antrum were detected by Wes-tern blot and real-time quantitative PCR, respectively. RESULTS: Compared with the normal group, the blood glucose, the apoptosis index, the protein and gene expressions of Caspase-3 and Bax were significantly increased (P<0.01), and the gastric emptying rate, intestinal propulsive rate, the protein and gene expressions of Bcl-2 were considerably decreased (P<0.01) in the model group. In contrast to the model group, the blood glucose in the EA, moxibustion and combination groups, the apoptosis index in the 4 treatment groups, as well as Caspase-3 protein, Bax protein and mRNA expressions in the medication, EA and combination groups, Caspase-3 protein and mRNA, Bax mRNA expressions in the moxibustion group were significantly decreased (P<0.01, P<0.05); while the gastric emptying rate and intestinal propulsive rate in the 4 treatment groups, and Bcl-2 protein and mRNA expressions in the medication and combination groups, Bcl-2 mRNA expressions in the EA and moxibustion groups were obviously increased (P<0.01). The effects of EA+moxibustion were significantly superior to those of simple EA, moxibustion or medication in increasing gastric emptying rate and intestinal propulsive rate, and in lowering blood glucose (P<0.05, P<0.01). And the effects of the combination treatment were better than those of EA in lowering Caspase-3 protein and Bax mRNA expressions (P<0.01), and in increasing Bcl-2 protein and mRNA expressions (P<0.05, P<0.01). Also the effects of the combination treatment were better than those of moxibustion in lowering the apoptosis index, Caspase-3 protein, and Bax protein and mRNA expressions (P<0.01, P<0.05), and in increasing Bcl-2 protein expression (P<0.05). CONCLUSION: EA combined with Zhuang-medicine-thread moxibustion can reduce blood glucose and improve gastrointestinal motility in DGP rats, which may be related to its effect in regulating of Caspase-3, Bax and Bcl-2 expression.

[Effect of ZhuLian's type â ¡ inhibition needling technique on endocrine metabolism in rats with polycystic ovary syndrome and insulin resistance].

OBJECTIVE: To observe the effect of ZhuLian's type Ⅱ inhibition needling technique on sex hormones, insulin resistance and ovarian morphology in rats with polycystic ovary syndrome (PCOS), so as to explore its mechanism on regulating endocrine and metabolic dysfunction of PCOS. METHODS: Twenty-four female SD rats were randomly divided into control, model and ZhuLian acupuncture groups (n=8 in each group). The PCOS model was established by continuous gavage of letrozole combined with high-fat diet. Rats of the ZhuLian acupuncture group were stimulated at "Guanyuan"(CV4) and bilateral "Guilai"(ST29), "Zusanli"(ST36), "Sanyinjiao"(SP6), and "Fenglong"(ST40) by ZhuLian's type Ⅱ inhibition needling technique for 30 min, continuously intervented for 28 days. The body weight was measured before and after modeling and after intervention. The ovarian volume was calculated. HE staining was used to observe the changes of ovarian histology. Plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and fasting insulin (FINS) were measured by ELISA. LH/FSH ratio and insulin resistance index (HOMA-IR) were calculated. RESULTS: After modeling, the rats' body weight and the ovarian volume were increased (P<0.05), and the ovaries showed polycystic changes in the model group compared with the control group. And the FINS level, HOMA-IR, LH content and LH/FSH ratio were significantly increased (P<0.05), while the FSH level was decreased (P<0.05). After the intervention, the rats' body weight and ovarian volume were decreased (P<0.05), and a small number of preantral follicles and more antral follicles were observed under microscope, and the levels of LH, LH/FSH, FINS and HOMA-IR were significantly decreased (P<0.05), while FSH content was increased (P<0.05) in the ZhuLian acupuncture group than in the model group. CONCLUSION: ZhuLian's type Ⅱ inhibition needling technique can improve the endocrine and metabolic disorders in PCOS rats, which may be related to the regulation of endocrine system.

[Efficacy and action mechanism of ZHU Lian inhibition type â acupuncture for acne with spleen-stomach dampness-heat].

OBJECTIVE: To observe the efficacy of ZHU Lian inhibition type Ⅰ acupuncture for acne with spleen-stomach dampness-heat, and to explore its possible action mechanism. METHODS: A total of 82 patients of acne with spleen-stomach dampness-heat were randomly divided into an observation group and a control group, 41 cases in each group. The patients in the control group were treated with danshentong capsules (1 g, 3 times per day) and 0.1% adapalene gel smear (once every night) for 4 weeks. The patients in the observation group were treated with ZHU Lian inhibition type Ⅰ acupuncture at Hegu (LI 4), Neiting (ST 44), Quchi (LI 11), Yangbai (GB 14), Sibai (ST 2), Qihai (CV 6), Xuehai (SP 10), Yinlingquan (SP 9) and skin lesions, once every other day, 7 times as a course of treatment, totaling for 2 courses of treatment. The skin lesion score of the global acne grading system (GAGS) and quality of life-acne (Qol-Acne) score as well as the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were compared between the two groups before and after treatment; the clinical efficacy were compared between the two groups, and the recurrence rate was evaluated 4 weeks after treatment. RESULTS: After treatment, the skin lesion scores of GAGS in the two groups were reduced (P<0.05), and the score in the observation group was lower than that in the control group (P<0.05). After treatment, the Qol-Acne scores were increased in the two groups (P<0.05), and the score in the observation group was higher than that in the control group (P<0.05). The serum levels of TNF-αand IL-6 in the two groups were reduced after treatment (P<0.05), and those in the observation group were lower than the control group (P<0.05). The total effective rate was 95.1% (39/41) in the observation group, which was higher than 82.9% (34/41) in the control group (P<0.05). Four weeks after treatment, the recurrence rate of acne lesions was 10.3% (4/39) in the observation group, which was lower than 32.4% (11/34) in the control group (P<0.05). CONCLUSION: ZHU Lian inhibition type Ⅰ acupuncture is effective for acne with spleen-stomach dampness-heat, and the recurrence rate is low. Its mechanism may be related to the reduction of serum inflammatory factor TNF-α and IL-6.

[Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on silent information regulator-1/nuclear factor κB signaling pathway in gastric antrum of diabetic gastroparesis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) combined with Zhuang-medicine-thread moxibustion on silent information regulator-1 (SIRT1)/nuclear factor kappa B (NF-κB) signal pathway and inflammatory factor expression in gastric antrum tissue of diabetic gastroparesis (DGP) rats, so as to explore its mechanism underlying improvement of DGP. METHODS: Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion (moxibustion) and EA+moxibustion groups (n=12 per group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension. EA (10 Hz /50 Hz, 2 mA) or moxibustion (3 cones) or EA+moxibustion was applied to "Zhongwan"(CV12), bilateral "Neiguan"(PC6) and bilateral "Sanyinjiao"(SP6) of the related group for 20 min, once a day for 3 weeks. Blood glucose, gastric emptying rate and intestinal propulsion rate were measured. The levels of serum interleukin (IL)-6, IL-8, IL-10 and tumor necrosis factor-α(TNF-α) were detected by ELISA; the phosphorylation level of the phosphorylated inhibitor of nuclear factor κBα inhibitor (pIκ-Bα), the protein and mRNA expression of NF-κB p65 and SIRT1 in the gastric antrum tissue were detected by Western blot and real-time quantifitative PCR, respectively. RESULTS: (1) Compared with the normal group, the levels of blood glucose, serum IL-6, IL-8, TNF-α, and gastric pIκ-Bα and NF-κB p65 protein and mRNA expressions were significantly increased (P<0.01), and the gastric emptying rate, intestinal propulsion rate, serum IL-10 level, and SIRT1 protein and mRNA expressions were considerably decreased in the model group (P<0.01). (2) In contrast to the model group, the blood glucose in the EA, moxibustion and EA+moxibustion groups, serum IL-6, IL-8 and TNF-α levels in the 4 treatment groups, as well as NF-κB p65 protein expression in the medication and EA+moxibustion groups, and NF-κB p65 mRNA expression and pIκ-Bα protein and mRNA expression in the 4 treatment groups were significantly decreased (P<0.01, P<0.05); while the gastric emptying rate and intestinal propulsive rate and IL-10 content in the 4 treatment groups, and SIRT1 protein and mRNA expression in the medication and EA+moxibustion groups were obviously increased (P<0.05, P<0.01). (3) The effects of EA+moxibustion were significantly superior to those of simple EA and moxibustion in increasing gastric emptying rate, IL-10, SIRT1 protein expression (P<0.05, P<0.01), and in lowering IL-8 and TNF-α contents, pIκ-Bα protein and mRNA expression and NF-κB p65 mRNA expression (P<0.05, P<0.01). No significant differences were found among the 4 intervention groups in promoting the intestinal propulsive rate and among the EA, moxibustion and EA+moxibustion groups in lowering blood glucose (P>0.05). CONCLUSION: EA combined with Zhuang-medicine-thread moxibustion can effectively reduce the level of serum inflammatory factors and regulate SIRT1/NF-κB signal pathway in DGP rats, which may contribute to its function in improving gastrointestinal movement.

Autophagy restricts mitochondrial DNA damage-induced release of ENDOG (endonuclease G) to regulate genome stability.

Genotoxic insult causes nuclear and mitochondrial DNA damages with macroautophagy/autophagy induction. The role of mitochondrial DNA (mtDNA) damage in the requirement of autophagy for nuclear DNA (nDNA) stability is unclear. Using site-specific DNA damage approaches, we show that specific nDNA damage alone does not require autophagy for repair unless in the presence of mtDNA damage. We provide evidence that after IR exposure-induced mtDNA and nDNA damages, autophagy suppression causes non-apoptotic mitochondrial permeability, by which mitochondrial ENDOG (endonuclease G) is released and translocated to nuclei to sustain nDNA damage in a TET (tet methylcytosine dioxygenase)-dependent manner. Furthermore, blocking lysosome function is sufficient to increase the amount of mtDNA leakage to the cytosol, accompanied by ENDOG-free mitochondrial puncta formation with concurrent ENDOG nuclear accumulation. We proposed that autophagy eliminates the mitochondria specified by mtDNA damage-driven mitochondrial permeability to prevent ENDOG-mediated genome instability. Finally, we showed that HBx, a hepatitis B viral protein capable of suppressing autophagy, also causes mitochondrial permeability-dependent ENDOG mis-localization in nuclei and is linked to hepatitis B virus (HBV)-mediated hepatocellular carcinoma development.Abbreviations: 3-MA: 3-methyladenine; 5-hmC: 5-hydroxymethylcytosine; ACTB: actin beta; ATG5: autophagy related 5; ATM: ATM serine/threonine kinase; DFFB/CAD: DNA fragmentation factor subunit beta; cmtDNA: cytosolic mitochondrial DNA; ConA: concanamycin A; CQ: chloroquine; CsA: cyclosporin A; Dox: doxycycline; DSB: double-strand break; ENDOG: endonuclease G; GFP: green fluorescent protein; Gy: gray; H2AX: H2A.X variant histone; HBV: hepatitis B virus; HBx: hepatitis B virus X protein; HCC: hepatocellular carcinoma; I-PpoI: intron-encoded endonuclease; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOMP: mitochondrial outer membrane permeability; mPTP: mitochondrial permeability transition pore; mtDNA: mitochondrial DNA; nDNA: nuclear DNA; 4-OHT: 4-hydroxytamoxifen; rDNA: ribosomal DNA; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TET: tet methylcytosine dioxygenase; TFAM: transcription factor A, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

[Effect of moxibustion with ignited Zhuang-medicine medicated-thread on interstitial cells of Cajal in gastric antrum in diabetic gastroparesis rats].

OBJECTIVE: To observe the effect of Zhuang-medicine medicated-thread moxibustion therapy on interstitial cells of Cajal (ICC) in the gastric antrum of diabetic gastroparesis (DGP) rats, so as to investigate its mechanism underlying improving DGP. METHODS: Sprague-Dawley (SD) male rats were randomly divided into 3 groups: control (n = 30) , model (n = 30) and moxibustion (n = 30). The DGP model was established by intraperitoneal injection of streptozotocin (STZ) and by feeding the rats with high fat-sugar forage. Zhuang-medicine medicated-thread moxibustion was applied to "Zhongwan" (CV 12), bilateral "Pishu" (BL 20), "Weishu" (BL 21), "Neiguan" (PC 6) and "Zusanli" (ST 36) once per day, for 3 weeks except weekends. The gastrointestinal propulsion rate and weight of stool in 24 h were determined, and c-kit (a marker for ICC) expression of the gastric antrum tissue was measured by immunohistochemistry. RESULTS: The stool weight was significantly higher in the model group than in the control group (P < 0.01), but the rate of gastrointestinal propulsion and the rate of c-kit immunoreaction (IR) positive cells in the gastric antrum tissue were significantly lower in the model group than in the control group (P < 0.01). After moxibustion, the increased stool weight and the decreased gastrointestinal propulsion rate and decreased c-kit IR-positive cell rate were reversed (P < 0.01). CONCLUSION: Zhuang-medicine medicated-thread moxibustion therapy can improve gastrointestinal function in DGP rats, which may be associated with its effect in up-regulating the expression of c-kit IR-positive ICC.

[Effect of drug-paste separated moxibustion on expression of estrogen, progestogen and their endometrial receptor mRNA in rats with primary dysmenorrhea].

OBJECTIVE: To observe the effect of drug-paste separated moxibustion of "Mingmen" (GV 4) on the levels of serum estrogen (E2) and progesterone (P) and their endometrial receptor mRNA expression in rats with primary dysmenorrhea in order to investigate its mechanism underlying improvement of primary dysmenorrhea. METHODS: A total of 100 female SD rats were randomized into control, model, medication, acupuncture and moxibustion groups, with 20 rats in each group. Primary dysmenorrhea model was established by subcutaneous injection of Benzestrofol for 10 days and intraperitoneal injection of Oxytocin for 1 d. Rats of the medication group were fed with extractum leonuri inspissatum (8 g/100 g) and those of the moxibustion group treated with drug-paste separated moxibustion at "Mingmen" (GV 4). For rats of the acupuncture group, a filiform needle was inserted into GV 4, manipulated for a while and retained for 30 min. The treatment of the latter 3 groups was conducted once daily for 7 days. The rat's body-writhing latency and times during 30 min were recorded. The contents of serum E2 and P were detected by ELISA, and the expression of estrogen receptor (ER) mRNA and progesterone receptor (PR) mRNA in the endometrium was determined by quantitative real-time (RT)-PCR. RESULTS: (1) The body-writhing latency was shorter and the writhing times were more in the model group than in the control group (P < 0.01). Compared with the model group, the body-writhing latency was significantly increased and the writhing times were obviously decreased in the medication, acupuncture and moxibustion groups (P < 0.01). There were no significant differences among the medication, acupuncture and moxibustion groups in the body-writhing latency (P > 0.05), but the body-writhing numbers of the acupuncture and moxibustion groups were markedly lower than that of the medication group (P < 0.01). (2) Compared with the control group, serum E2 content and endometrial ER mRNA expression level were significantly increased, and serum P content and endometrial PR mRNA level evidently decreased in the model group (P < 0.01, P < 0.05). In comparison with the model group, serum E2 contents and endometrial ER mRNA expression levels were considerably down-regulated, and serum P contents and endometrial PR mRNA expression levels markedly up-regulated in the medication, acupuncture and moxibustion groups (P < 0.01, P < 0.05). The effects of the moxibustion group were significantly superior to those of the acupuncture and medication groups, and those of the acupuncture group were also significantly superior to those of the medication group in lowering E2 and endometrial ER mRNA levels, and raising serum P and endometrial PR mRNA expression levels (P < 0.01, P < 0.05). CONCLUSION: Drug-paste separated moxibustion of GV 4 is effective in relieving pain in primary dysmenorrheal rats, which is probably associated with its effects in down-regulating serum E2 content and endometrial ER mRNA expression, and up-regulating serum P and endometrial PR mRNA expression levels.