[Synergistic effect of acupuncture and moxibustion on expression of renal Fas and FasL mRNAs and proteins in contrast-induced nephropathy diabetic rats].

OBJECTIVE: To observe the effect of acupuncture and moxibustion on renal Fas and Fas ligand (FasL) expression in contrast-induced nephropathy (CIN) diabetic rats, so as to explore its protective mechanisms. METHODS: A total of 40 diabetic male SD rats were randomly divided into control, model, acupuncture, moxibustion and combined treatment (acupuncture combined with moxibustion) groups (n＝8 rats in each group). The diabetic model was established by i．p. injection of Streptozotocin (60 mg/kg), followed by regular raising for 9 weeks. Then, the manual acupuncture, moxibustion or acupuncture plus moxibustion at "Sanyinjiao"(SP6) "Shenshu"(BL23) and "Pishu"(BL20) was given to the diabetic rats, once daily for 7 days. On the 8th day, these diabetic rats received i．p. injection of compound Meglumine Diatrizoate Injection (10 mL/kg) for establishing CIN model. Then, the venous blood specimens were collected for detecting contents of serum urine nitrogen (BUN, with urease method) and creatinine (Scr, with picric acid method). The kindey tissues were exteriorized for detecting nitricoxide synthase (NOS, with oxidation-reduction method), malondialdehyde (MDA, with thiobarbituric acid method), superoxide dismutase (SOD, with xanthine oxidase technique) and total antioxidant capacity (T-AOC, with colorimetric technique). The expression levels of Fas and FasL in the kidney tissues were detected by quantitative real-time PCR and Western blot separately. The histopathological and ultrastructural changes of the kidney were observed under microscope (after H．E. staining) and transmission electron microscope, respectively. RESULTS: Following modeling and compared with the control group, the BUN, Scr and renal MDA contents, and Fas and FasL mRNA and protein expression levels were significantly increased (P<0.01, P<0.05), and renal NOS, SOD and T-AOC levels considerably decreased in the model group (P<0.05). After the interventions and compared with the model group, serum BUN and Scr contents and renal Fas mRNA and protein expression levels in both acupuncture group and combined treatment group, BUN in the moxibustion group, and renal MDA content and FasL mRNA and protein expression levels in the 3 treatment groups were obviously down-regulated (P<0.05，P<0.01), while renal NOS in the 3 treatment groups, and SOD and T-AOC levels in the combined treatment group were considerably up-regulated (P<0.01，P<0.05). The effects of the combined treatment were significantly superior to those of simple acupuncure and simple moxibustion in down-regulating serum BUN contents, and Fas and FasL mRNA and protein expression levels, and in up-regulating renal NOS, SOD and T-AOC activity (P<0.05). No significant differences were found between the acupuncture and moxibustion groups in down-regulating serum BUN, Scr and renal MDA contents, and Fas and FasL mRNA and protein levels, as well as in up-regulating NOS content (P>0.05). H．E. staining showed lobular changes of glomeruli, many local necrosis and vascular degeneration in the model group, and transmission electron microscope displayed thickened basement membrane of the glomerular blood loop, many vacuoles in the cytoplasm of renal tubular epithelial cells, severely swollen mitochondria with disordered, broken or disappeared cristae, and some apoptotic cells in the model group, which were relatively milder in the combined treatment group. CONCLUSION: Joint application of acupuncture and moxibustion can reduce the oxidative stress and renal injury in CIN diabetic rats, which may be associated with its function in down-regulating the expression of renal Fas and FasL genes and proteins. Acupuncture and moxibustion has a synergistic effect.

[Electroacupuncture of Multiple Acupoints Is Significantly Superior to That of Single Acupoint in Improving Sleep by Regulating Serum Sleep-promoting and Awakening-promoting Factors in Insomnia Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of single and multiple acupoints on sleep and concentrations of interlukin-1 ß(IL-1 ß), brain-derived neurotrophic factor (BDNF), prostaglandin D2(PGD2) and melatonin (MLT, sleep-promoting factors) and corticosterone (CORT, awakening-promoting factor) in the serum in insomnia rats, so as to explore its efficacy difference and the mechanism underlying improving sleep. METHODS: Fifty-four male SD rats were randomly divided into control, model, EA-Baihui (GV 20), EA-Shenmen (HT 7), EA-Sanyinjiao (SP 6) and EA-GV 20＋HT 7＋SP 6 groups (n＝9 rats in each group). The insomnia model was established by intraperitoneal injection of para-chlorophenylalanine (PCPA, 300 mg/kg) once daily for 2 days. In the EA-GV 20, EA-HT 7, EA-SP 6 and EA-GV 20＋HT 7＋SP 6 groups, EA stimulation was administrated for 30 min, once a day for 4 days. The sleep onset latency and sleep duration were measured after intraperitoneal injection of pentobarbital sodium (35 mg/kg). The concentrations of IL-1 ß， BDNF, MLT, PGD2and CORT in the serum were detected by ELISA. RESULTS: After EA stimulation of GV 20, HT 7, SP 6 and GV 20＋HT 7＋SP 6, the sleep latency was significantly shortened (P<0.05, P<0.01, except SP 6), and the sleep duration was remarkably prolonged in comparison with the model group (P<0.05, P<0.01), and the therapeutic effects of EA-GV 20＋HT 7＋SP 6 were significantly superior to those of EA-GV 20, EA-HT 7 and EA-SP 6 in shortening the sleep latency and lengthening the sleep duration (P<0.05). Following modeling, the concentrations of IL-1 ß， BDNF, PGD2 and MLT were significantly down-regulated, and the CORT level was markedly up-regulated in the model group relevant to the control group (P<0.05). Following EA，modeling induced dramatic decrease of serum IL-1 ß， BDNF, PGD2 and MLT was considerably up-regulated, and the increased CORT level markedly down-regulated in the EA-GV 20, EA-HT 7, EA-SP 6 and EA-GV 20＋HT 7＋SP 6 groups (P<0.05). The effects of EA-GV 20＋HT 7＋SP 6 were evidently superior to those of EA-GV 20 and EA-SP 6 in up-regulating serum IL-1 ß， BDNF and PGD2levels, and to those of HT 7, GV 20 and SP 6 in up-regulating serum MLT level, and significantly superior to those of EA-ST 7 and EA-SP 6 in down-regulating serum CORT (P<0.05). CONCLUSION: EA stimulation of HT 7, GV 20, SP 6 and GV 20＋HT 7＋ SP 6 can significantly improve the sleep in insomnia rats, which is closely associated with its effects in regulating serum sleep-promoting factors and awakening-promoting factor. Joint administration of EA of GV 20＋HT 7＋ SP 6 has a better effect than the single acupoint mentioned above.

[Transcutaneous Electrostimulation of Auricular Otopoints Reduces Epileptic Attack Possibly by Suppressing Hippocampal Gliocyte Proliferation and Regulating IL-6 and IL-10 Expression in Chronic Temporal Lobe Epilepsy Rats].

OBJECTIVE: To observe the effect of transcutaneous otopoint electrostimulaiton (TCOES) on seizure frequency, immunoreactivity of hippocampal gliocytes and expression of proinflammatory cytokine interleukin-6(IL-6) and anti-inflammatory cytokine IL-10 in chronic temporal lobe epilepsy (CTLE) rats, so as to investigate its antiepileptic mechanism. METHODS: Thirty-six SD rats were randomly divided into control, model and TCOES groups (n=12 in each group). The CTLE model was established by intraperitoneal injection (i.p.i.) of lithium chloride (127.2 mg/kg), scopolamine (1 mg/kg, 20 h after the 1st injection) and pilocarpine (10 mg/kg, 30 min after scopolamine injection). Rats of the control group were treated by i.p.i. of normal saline. TCOES (1 mA, 20 Hz) was applied to bilateral otopoint "Heart"-"Lung"-"Subcortex" region for 20 min, once daily for 6 weeks. The epileptic attack was observed by a video monitoring system. The numbers of ionized calcium-binding adapter molecule-1 (Iba 1)-labeled microgliacytes and glial fibrillary acidic protein (GFAP)-labeled astrocytes in the CA 1 and CA 3 regions of hippocampus were counted under light microscope after immunostaining, and the expression levels of hippocampal IL-6 and IL-10 proteins and genes were determined by immunofluorescence and quantitative real-time PCR, respectively. RESULTS: After TCOES intervention, the seizure frequency was significantly decreased in comparison with pre-treatment(P<0.05), modeling-induced dramatic increase of the numbers of microgliacytes and astrocytes,IL-6 immunoactivity in the hippocampal CA 1 and CA 3 regions, and IL-6 mRNA expression in the hippocampus were significantly suppressed (P<0.05), and hippo-campal IL-10 immunoactivity and mRNA expression were considerably up-regulated in comparison with the model group (P<0.05). CONCLUSIONS: TCOES intervention has an antiepileptic effect in CTLE rats, which may be associated with its effects in suppressing gliocyte proliferation, suppressing the expression of proinflammatory cytokine IL-6, and up-regulaiting the expression of anti-inflammatory cytokine IL-10 in the hippocampus.

[Effects of Electroacupuncture on Activities of Satellite Glial Cells of Dorsal Root Ganglia in Rats with Neck Incision Pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Futu"(LI 18), etc. on activities of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) in rats with neck-incision pain so as to explore its mechanism underlying reduction of post-surgical pain of thyroidectomy. METHODS: Male SD rats were randomly divided into control, model, EA-Futu (LI 18), EA-Hegu (LI 4)-Neiguan (PC 6), and EA-Zusanli (ST 36)-Yanglingquan (GB 34) groups, with 20 rats in each group. The neck-incision pain model was established by making a longitudinal incision and repeated mechanical stimulation. In the EA-LI 18, EA-LI 4-PC 6 and EA-ST 36-GB 34 groups, EA stimulation was administrated for 30 min, once a day,continuously for 3 days. The thermal pain threshold (PT) of the neck-incision region was detected. The immunoactivity of glial fibrillary acidic protein (GFAP,a specific marker for SGCs) and connexin 43 (Cx 43) of DRGs (C 2-C 6) was determined by fluorescent immunohistochemistry. The expression levels of GFAP, IL-1 ß, IL-6, and TNF-α mRNAs were determined by quantitative Real-time PCR, and the contents of IL-1 ß,IL-6,TNF-α assayed by enzyme linked immunosorbent assay (ELISA) and the expression of Cx 43 protein was detected by Western blot. RESULTS: After EA intervention at LI 18 and LI 4-PC 6 (but not ST 36-GB 34), neck incision-induced reduction of the thermal PT was obviously prolonged in comparison with the model group (P<0.05),suggesting a pain relief. The expression levels of GFAP, IL-1 ß, IL-6 and TNF-α mRNAs and Cx 43 protein, and the contents of IL-1 ß, IL-6 and TNF-α in C 2-C 6 DRGs were all significantly up-regulated in the model group relevant to those of the control group (P<0.05). Following EA, modeling induced dramatic increase of expression of GFAP, IL-1 ß, IL-6 and TNF-α mRNAs and Cx 43 protein in both EA-LI 18 and EA-LI 4-PC 6 groups, and the contents of IL-1 ß and TNF-α in the EA-LI 18 group, IL-6 in the EA-LI 4-PC 6 group was considerably down-regulated (P<0.05). In comparison with the model group, no significant changes were found in all the abovementioned indexes of EA-ST 36 -GB 34 group except the down-regulated IL-1 ß and TNF-α mRNAs, in the contents of IL-1 ß and TNF-α of the EA-LI 4-PC 6 group, and in the IL-6 content of the EA-LI 18 group (P>0.05). CONCLUSIONS: EA stimulation of LI 18 and LI 4-PC 6 can significantly suppress pain reaction of neck incision in the rat, which is closely associated with its effects in down-regulating the activity of SGCs, decreasing the release of pro-inflammatory cytokines and in weakening the expression of Cx 43 in the cervical DRGs.

Angelica polysaccharides inhibit the growth and promote the apoptosis of U251 glioma cells in vitro and in vivo.

BACKGROUND: Angelica sinensis (Oliv) Diels (Apiaceae) is a traditional medicine that has been used for more than 2000 years in China. It exhibits various therapeutic effects including neuroprotective, anti-oxidant, anti-inflammatory, and immunomodulatory activities. Angelica polysaccharides (APs), bioactive constituents of Angelica have been shown to be responsible for these effects; however, the utility of APs for the treatment of glioma and their mechanism of action remain to be elucidated. PURPOSE: In this study, we investigated the inhibitory effects of APs on a glioma cell line and their molecular mechanism of action. STUDY DESIGN: U251 cells were utilized to confirm the effects of APs on glioma. METHODS: The human glioblastoma cell line U251 was utilized for both in vitro and in vivo models, in which we tested the effects of APs. Flow cytometry, gene expression analysis, western blotting, and MTT assays were used to elucidate the effects of APs on cell proliferation, cell cycle, and apoptosis. RESULTS: The results demonstrated that APs significantly inhibited the growth and proliferation of U251 cells and induced their apoptosis. Furthermore, APs effectively reduced the expression of several cell cycle regulators: cyclins D1, B, and E. The apoptosis suppressor protein Bcl-2 was also downregulated, and the expression of pro-apoptotic proteins Bax and cleaved-caspase-3 increased. Additionally, APs inhibited the transforming growth factor (TGF)-ß signaling pathway and stimulated the expression of E-cadherin, thus prohibiting cell growth. CONCLUSION: In conclusion, the results indicate that APs attenuate the tumorigenicity of glioma cells and promote their apoptosis by suppressing the TGF-ß signaling pathway. The present study therefore provides evidence of the inhibitory effects of APs against glioma progression, and proposes their potential application as alternative therapeutic agents for glioma.