Long-read bitter gourd (Momordica charantia) genome and the genomic architecture of nonclassic domestication.

The genetic architecture of quantitative traits is determined by both Mendelian and polygenic factors, yet classic examples of plant domestication focused on selective sweep of newly mutated Mendelian genes. Here we report the chromosome-level genome assembly and the genomic investigation of a nonclassic domestication example, bitter gourd (Momordica charantia), an important Asian vegetable and medicinal plant of the family Cucurbitaceae. Population resequencing revealed the divergence between wild and South Asian cultivars about 6,000 y ago, followed by the separation of the Southeast Asian cultivars about 800 y ago, with the latter exhibiting more extreme trait divergence from wild progenitors and stronger signs of selection on fruit traits. Unlike some crops where the largest phenotypic changes and traces of selection happened between wild and cultivar groups, in bitter gourd large differences exist between two regional cultivar groups, likely reflecting the distinct consumer preferences in different countries. Despite breeding efforts toward increasing female flower proportion, a gynoecy locus exhibits complex patterns of balanced polymorphism among haplogroups, with potential signs of selective sweep within haplogroups likely reflecting artificial selection and introgression from cultivars back to wild accessions. Our study highlights the importance to investigate such nonclassic example of domestication showing signs of balancing selection and polygenic trait architecture in addition to classic selective sweep in Mendelian factors.

Assessment of Genetic Differentiation and Linkage Disequilibrium in Solanum pimpinellifolium Using Genome-Wide High-Density SNP Markers.

To mine new favorable alleles for tomato breeding, we investigated the feasibility of utilizing Solanum pimpinellifolium as a diverse panel of genome-wide association study through the restriction site-associated DNA sequencing technique. Previous attempts to conduct genome-wide association studies using S. pimpinellifolium were impeded by an inability to correct for population stratification and by lack of high-density markers to address the issue of rapid linkage disequilibrium decay. In the current study, a set of 24,330 SNPs was identified using 99 S. pimpinellifolium accessions from the Tomato Genetic Resource Center. Approximately 84% of PstI site-associated DNA sequencing regions were located in the euchromatic regions, resulting in the tagging of most SNPs on or near genes. Our genotypic data suggested that S. pimpinellifolium were divided into three single-ancestry subpopulations and four mixed-ancestry subpopulations. Additionally, our SNP genotypic data consistently confirmed the genetic differentiation, achieving a relatively reliable correction of population stratification. Previous studies utilized the 8K tomato SNP array, SolCAP, to investigate the genetic variation of S. pimpinellifolium and we performed a meta-analysis of these genotypes. The result suggested SolCAP array was less appropriate to profile the genetic differentiation of S. pimpinellifolium when more accessions were involved because the samples belonging to the same accession demonstrated different genome patterns. Moreover, as expected, rapid linkage disequilibrium decay was observed in S. pimpinellifolium, especially in euchromatic regions. Approximately two-thirds of the flanking SNP markers did not display linkage disequilibrium based on r2 = 0.1. However, the 18-Kb linkage disequilibrium decay indeed reveals the potential of single-gene resolution in GWAS when markers are saturated.

[Effect of Electroacupuncture Intervention on Gastrointestinal Motility and Expression of Insulin-like Growth Factor 1 and Its Receptor Proteins in Gastric Antrum in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention of "Zusanli"(ST 36), etc. on gastrointestinal motility and levels of insulin-like growth factor 1 (IGF-1) and IGF-1 receptor (IGF-1 R) proteins in the gastric antrum in diabetic gastroparesis (DGP) rats, so as to study its mechanism underlying improvement of DGP. METHODS: Fifty SD rats, half male and half female, were randomly allocated to control, model, EA acupoint, EA non-acupoint and medication (metoclopramide) groups (n=10 rats/group). The DGP model was induced by 2% streptozotocin (STZ, i.p.i.,55 mg/kg) and high fat-sugar forage for 8 weeks. EA (10 Hz/50 Hz, 2 mA) was applied to "Zusanli"(ST 36), "Liangmen"(ST 21), "Sanyinjiao"(SP 6) and non-acupoints (about 5 mm lateral to ST 36, ST 21 and SP 6, respectively) for 20 min, one daily for 15 days, and 1.7% metocloppramide (1 mL/100 g) was given (gavage) to rats of the medication group, once daily for 15 days. After the treatment, the rats' general conditions were scored, the blood glucose level was determined using a glucometer, and the gastrointestinal mobility was evaluated by measuring the gastric emptying rate (GER) and the intestinal propulsion rates (IPR) marked by oral-infused phenol red solution (50 mg/dL). The immunoactivity levels of IGF-1 and IGF-1 R of gastric antrum tissue were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: After modeling, the symptom integrative score, the blood glucose, IGF-1 and IGF-1 R levels were significantly increased (P<0.01), and the GER and IPR were notably decreased relevant to the control group (P<0.01). After EA stimulation of ST 36, ST 21 and SP 6, the symptom score, blood glucose, and IGF-1 and IGF-1 R levels of the gastric antrum were significantly reduced, while the GER and IPR were considerably increased (P<0.05,P<0.01). Except the GER and IPR were considerably increased in the medication group(P<0.05,P<0.01), no significant changes were found in the symptom integrative score, the blood glucose, IGF-1 and IGF-1 R levels in both EA non-acupoint and medication groups relevant to the model group (P>0.05),the GER and IPR in the EA non-acupoint group (P>0.05). Compared with the EA acupoint group, the symptom integrative score, IGF-1 and IGF-1 R levels were significantly increased in both EA non-acupoint and medication groups(P<0.05,P<0.01). CONCLUSIONS: EA stimulation can improve gastrointestinal motility and lower blood glucose in DGP rats, which may be closely associated with its effectiveness in reducing IGF-1 and IGF-1 R immunoactivity levels in gastric antrum.

[Effects of Electroacupuncture on Ultrastructure of Interstitial Cells of Cajal and Stem Cell Factor-kit Signal Pathway of Gastric Antrum in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on gastrointestinal motility and the ultrastructure of interstitial cells of Cajal (ICC) and the expressions of c-kit receptor protein and stem cell factor (SCF) mRNA in diabetic gastroparesis (DGP) rats, so as to explore its mechanism. METHODS: Fifty SD rats were randomly divided into normal, model, acupoint, non-acupoint and metoclopramide groups (n＝10 rats/group). DGP model was established by intraperitoneal injection of streptozotocin (STZ, 2%), and raised with high-sugar high-fat diet irregularly. EA (sparse-dense, 10 Hz/50 Hz, 2 mA, 20 min) was applied at "Zusanli" (ST 36), "Liangmen" (ST 21) and "Sanyinjiao" (SP 6), and the corresponding non-acupoints of the 3 acupoints, daily for 15 days. The rats in metoclopramide group received intragastric administration of metoclopramide (1.7%, 1 mL/100 g) for 15 days, once a day. Blood sugar was determined with One Touch blood glucose test paper. The gastric emptying rate (GER) and the intestinal propulsion rate (IPR) were measured by intragastric phenol red. The ultrastructure of ICC was detected by transmission electron microscopy. The expression levels of c-kit receptor protein and SCF mRNA of gastric antrum were examined respectively by Western blot and RT-PCR. RESULTS: Compared with the normal group, the blood glucose significantly increased in the model group (P<0.01), while the GER, IRP and the expression level of SCF mRNA in the gastric antrum significantly decreased (P<0.01), and the ultrastructure of ICC appeared apoptosis-like changes. The blood glucose of the EA group was obviously decreased compared with that of the model group (P<0.05); the GER and IRP significantly increased(P<0.05, P<0.01); the expression level of SCF mRNA increased (P<0.01), the number of ICC increased and its ultrastructure was repaired. There was some relief on ICC ultrastructure in the acupoint group compared with that in the non-acupoint group; and SCF mRNA increased (P<0.05). There was no significant difference on c-kit receptor expression among all the modeling groups (P＞0.05). CONCLUSIONS: EA at ST 36, etc. can regulate the blood glucose and improve gastrointestinal emptying in DGP rats. The mechanism may be related to up-regulating SCF mRNA, repairing ICC ultrastructure, restoring the pacing function, and improving gastrointestinal motility.

[Effect of Electroacupuncture at "Zusanli" (ST 36), etc. on Gastrointestinal Motility and Expression of Ghrelin mRNA and Growth Hormone Secretagogue Receptor mRNA in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Zusanli" (ST 36), "Liangmen" (ST 21) and "Sanyinjiao" (SP 6) on gastrointestinal motility and expression of ghrelin and growth hormone secretagogue receptor (GHSR) mRNA in diabetic gastroparesis (diabetic gastroparesis, DGP) rats, so as to explore its mechanism underlying improvement of diabetic gastroparesis. METHODS: Sixty SD rats were randomly and equally divided into normal control, model, EA acupoint, EA non-acupoint, medication (metoclopramide) groups (n = 12 rat/group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ, 55 mg/kg, 2%) and raised by high-sugar and high-fat fodder irregularly for 8 weeks. EA was applied to "Zusanli" (ST 36), "Liangmen" (ST 21) and "Sanyinjiao" (SP 6), and non-acupoints (about 5 mm lateral to the 3 acupoints, respectively). Those rats of the medication group were given with metoclopramide (1 mL/100 g). The blood sugar level was measured by glucometer, and the urine sugar level measured by Tes-Tape [urine glucose test paper]. The intestinal propulsion rate (IPR) was identified as ratio of the phenolsulfonphthalein promoting distance and the total length of the small intestine, and the gastric emptying rate (GER) was assessed by gavage of a liquid test meal (1.5 mL) containing a nonabsorbable marker (0. 5 mg/mL of phenol red in 5% glucose solution) and identification after being sacrificed 72 h after modeling. The expression levels of ghrelin mRNA and GHSR mRNA of gastric antrum tissue were examined by Quantitative real-time PCR. RESULTS: In comparison with the normal control group, the blood sugar and urine sugar levels of the model group was significantly increased in the model group (P < 0.01), while the GER and IPR and the expression level of ghrelin mRNA and GHSR mRNA in the gastric antrum were significantly decreased in the model group (P < 0.01, P < 0.05). Following EA stimulation of acupoint, the blood sugar and urine sugar levels were obviously down-regulated (P < 0.05), both GER and IPR, and ghrelin mRNA and GHSR mRNA expression levels in the EA-acupoint group were significantly up-regulated (P < 0.05, P < 0.01). No significant changes were found in both blood sugar and urine sugar levels, in both GER and IPR, and ghrelin mRNA and GHSR mRNA expression levels of both EA non-acupoint and medication groups after the intervention (P > 0.05). The GER and IPR were significantly higher in the EA acupoint group than in the EA non-acupoint group (P < 0.05, P < 0.01). CONCLUSION: EA stimulation of ST 36, ST 21 and SP 6 can promote the gastrointestinal motility of DGP rats, which may be closely associated with its effects in up-regulating the expression level of ghrelin mRNA and GHSR mRNA in gastric antrum.

[Serumimmunological study of moxibustion on helicobacter pylori gastritis in rats].

OBJECTIVE: To explore the immune mechanism of moxibustion on protecting gastric mucosa injury. METHODS: Forty healthy SD rats were randomly divided into four groups: a blank group, a model group, a moxibustion acupoint group and a moxibustion non-acupoint group, 10 rats in each one. Eight days before model establishment, moxibustion at "Zusanli" (ST 36), "Zhongwan" (CV 12), "Guanyuan" (CV 4), "Pishu" (BL 20) and "Weishu" (BL 21) was applied in the moxibustion acupoint group while these acupoints' controlled points were selected in the moxibustion non-acupoint group, and no treatment was given in the model group, once a day in three groups for continuous 16 days. The helicobacter pylori (Hp) model was established by intragastric administration of Hp. HE staining microscopic examination was used to observe inflammation severity in gastric mucosa, and enzyme-linked immunosorbent assay (ELISA) was adapted to measure content of heat shock protein (HSP) 72, TNF-alpha and IL-1beta, and real-time quantitative PCR was used to measure the expression of TLR2 mRNA, TLR4 mRNA, CD14 mRNA and MyD88 mRNA in peripheral blood mononuclear cells, and western blot method was used to measure content of NFkappaB and IkappaBalpha in peripheral blood mononuclear cells. RESULTS: Compared with the blank group, the expression of HP could be seen in the smear of gastric mucosa by Gram's staining in the model group; the inflammation severity score was obviously increased as well as content of serum HSP 72 and TNF-alpha and IL-1beta in gastric tissue; and expression of TLR2, 4 mRNA, CD14 mRNA, MyD88 mRNA, NFkappaB was increased (P < 0.01), but the expression of IkappaBalpha was reduced (P < 0.05). After the moxibustion, the inflammation severity score was reduced in the moxibustion acupoint group, and the content of serum HSP 72 was increased, and the expression of TNF-alpha and IL-1beta in gastric tissue and expression of TLR2 mRNA, TLR4 mRNA, CD14 mRNA, MyD88 mRNA and NFkappaB were reduced (P < 0.01), but the expression of IkappaBalpha was increased (P < 0.05). The differences between the moxibustion non-acupoint group and the model group were not significant (P > 0.05). CONCLUSION: The pretreatment of moxibustion at acupoints could induce the over expression of serum HSP 72. By combining TLR 2 and 4 receptors to trigger receptor signal transduction pathways, the releases of downstream signal substances are regulated; as a result, the releases of related immune substances are regulated to relieve the gastric mucosa injury of rats with HP gastritis.

[Study on release kinetics for gel of Aconitum brachypodum based on nonlinear mixed effect model].

OBJECTIVE: To establish the analytical method for the release kinetic (RK) of Aconitum Brachypodum gel based on the nonlinear mixed effect model (NLMEM), in order to rationally evaluate the drug release process and explain the release mechanism. METHOD: The zero-order kinetic model containing for non-corroded drug system with the random effect was taken as the base model. The fixed effect and random effect factors impacting the drug release were analyzed by PROC NLMIXED of SAS to establish the final typical model. Subsequently, 10 training subsets were randomly extracted from the primary data to respectively their RK models, calculate the corresponding predicted root-mean-square error and average relative error, and evaluate the model stability and prediction accuracy. RESULT: The burst effect F0 had a very significant effect on the RK model. Among the component factors, carbopol 940 showed an obvious effect on the inherence release speed constant k0 and the concentration gradient change constant a, with different variations on the basis of dosage range. The random effect factors of k0 and a had a significant impact. The final RK model was proved to be stable, effective and reliable in the cross validation. CONCLUSION: The drug release kinetic analysis method could be used to rationally evaluate the drug release process and explain the release mechanisms.

[Efficacy observation on acupuncture for essential hypertension of yin deficiency due to yang hyperactivity pattern].

OBJECTIVE: To observe the clinical effects of essential hypertension treated with acupuncture at Siguan [Hegu (LI 4) and Taichong (LR 3)], Quchi (LI 11) and Xingjian (LR 2). METHODS: Sixty cases of essential hypertension were randomly divided into an acupuncture group and a medication group, 30 cases in each group. Two groups of acupoint, which were (1) Siguan [Hegu (LI 4) and Taichong (LR 3)] and (2) Quchi (LI 11), Xingjian (LR 2), were selected alternatively in the acupuncture group, once everyday. In the medication group, captopril was prescribed for oral administration 25 mg per time, three times per day. Seven days made one session in both groups and totally three sessions were required. RESULTS: (1) After treatment, the blood pressure were decreased significantly in the two groups (all P < 0.01). Compared with the medication group, the diastolic pressure after 14 days, 21 days of treatment in the acupuncture group was decreased significantly (both P < 0.01). (2) In the terms of TCM syndrome score, the improvement of dizziness, soreness and weakness of waist and knees, palpitations was significant in the acupuncture group compared with that in the medication group(all P < 0.05). (3) The acupuncture group had less adverse reactions compared with the control group (P < 0.05). CONCLUSION: Acupuncture at Hegu (LI4) and Taichong (LR 3), Quchi (LI 11) and Xingjian (LR 2) can effectively decrease blood pressure; the effect of controlling diastolic pressure is better than captopril. It can also improve the symptoms of dizziness, soreness and weakness of waist and knees, and palpitations.

[Effect of manual acupuncture stimulation of "Zusanli" (ST 36) on gastric motility, and SP and motilin activities in gastric antrum and nucleus raphe magnus in gastric hyperactivity and hypoactivity rats].

OBJECTIVE: To observe the changes of gastric motility and levels of substance P (SP) and motilin (MTL) in the gastric antrum and Nucleus Raphe Magnus (NRM) after manual acupuncture stimulation of "Zusanli" (ST 36) in gastric hyperactivity and hypoactivity rats, so as to analyze the role of NRM in acupuncture mediated adjustment of gastric motility. METHODS: Fifty SD rats were randomly and equally divided into control, gastric hyperactivity (G-Hypera) model, gastric hypoactivity (G-Hypoa) model, acupuncture + G-Hypera and acupuncture + G-Hypoa groups (10 rats/group). G-Hypera model was established by intravenous (tail vein) injection of Maxolon (0.5 mL/200 g) and G-Hypoa model established by intravenous injection of Atropin (0.5 mL/200 g), respectively. After insertion of acupuncture needles into bilateral "Zusanli" (ST 36), the needles were repeatedly manipulated at a frequency of about 2 Hz for 5 min. The intragastric pressure was recorded and analyzed using a physiological signal analysis system. The SP and MTL contents of gastric antrum were measured by ELISA, and SP and MTL immunoactivity of NRM was determined by immunohistochemistry. RESULTS: In gastric hyperactivity rats, compared with the control group, the intragastric pressure (not systolic frequency), SP and MTL contents in the gastric antrum and MTL immunoactivity of NRM were significantly increased (P < 0.05, P < 0.01), while SP immunoactivity of NRM was apparently decreased in the G-Hypera model group (P < 0.01). After acupuncture stimulation, the intragastric pressure, gastric SP and MTL contents, and MTL immunoactivity of NRM were all significantly down-regulated (P < 0.05, P < 0.01), and SP immunoactivity of NRM was markedly up-regulated (P < 0.01) in comparison with the model group. In gastric hypoactivity rats, compared with the control group, the intragastric pressure and gastric systolic frequency, gastric SP and MTL contents, and MTL immunoactivity of NRM were significantly decreased (P < 0.05, PF < 0.01), while SP immunoactivity of NRM was remarkably up-regulated in the G-Hypoa model group (P < 0.01). After acupuncture stimulation, the above-mentioned 6 indexes were reversed significantly (P < 0.05, P < 0.01) in comparison with those of the model group. CONCLUSION: Acupuncture stimulation of "Zusanli" (ST 36) has an obvious bi-directional adjustment effect on gastric motility, which is closely associated with its functions in regulating gastric SP and MTL level and the expression of MTL and SP in the NRM of brainstem.

[Clinical observation on therapeutic effects of mild moxibustion in superficial gastritis patients with spleen-stomach deficiency-cold pattern].

OBJECTIVE: To observe the curative effect of different duration of mild moxibustion in superficial gastritis patients with deficiency cold pattern of spleen and stomach. METHODS: A total of 84 superficial gastritis patients were randomly divided into moxibustion-20 min group, moxibustion-40 min group and medication (Ranitidine) group, with 28 cases in each group. Mild moxibustion was applied to bilateral Zusanli (ST 36) and Zhongwan (CV 12, odd days), Pishu (BL 20) and Weishu (BL 21, even-numbered days) for 20 min and 40 min respectively, once daily for 20 days. The symptom integrative score of superficial gastritis patients and the therapeutic effects were assessed according to "The Scoring Table for Symptoms and Signs of Gastrointestinal Disorders of Chinese Medicine", etc. RESULTS: Following 20 days' treatment, of the three 28 gastrititis patients in the medication, moxibustion-20 min and moxibustion-40 min groups, 0, 1, and 0 cases were cured, 5, 7 and 12 experienced marked improvement in their symptoms, 15, 16 and 12 had an improvement, and 8, 4 and 4 were invalid, with the effective rates being 71.4%, 85.7% and 85.7%, respectively. After one month's follow up, of the three 28 cases in the medication, moxibustion-20 min and moxibustion-40 min groups, 0, 4 and 0 cases were cured, 4, 8, and 13 experienced marked improvement, 15, 16 and 13 had an improvement, 9, 0 and 2 were ineffective, with the effective rates being 67.9%, 100% and 92.9%, respectively. Self-comparison of each group showed that the integrative scores of symptoms were all decreased significantly after the treatment and one-month's follow up in the three groups (P < 0.01). The effective rates of both moxibustion-20 min and -40 min were significantly higher than that of the medication group after one month's follow up (P < 0.01, P < 0.05). The therapeutic effect of moxibustion-20 min group was significantly superior to that of the medication group in lowering the integrative score of symptoms after one month's follow up (P < 0.01). No significant differences were found among the three groups in the therapeutic effects and in reducing integrative scores of symptoms after 20 days' treatment (P > 0.05). CONCLUSION: Mild moxibustion is significantly superior to medication in long-term therapeutic effects in the treatment of superficial gastritis patients with spleen-stomach deficiency-cold pattern.

[Effect of moxibustion on motility, absorption and content of ATP in small intestine of spleen-deficiency rats].

OBJECTIVE: To explore the mechanism of reinforcing function of moxibustion to spleen-stomach. METHODS: Forty healthy Sprague Dawley rats were randomly divided into 4 groups: group A (blank group), group B (model group), group C (moxibustion group) and group D (herbs group). The rat model of spleen-deficiency was established by intragastric administration with 200% Dahuang (Rhubarb) infusion. The rats in group A and B, and D served as the blank control, model, and Sijunzi decoction group respectively, while those in group C received moxibustion at "Zusanli" (ST 36), "Zhongwan" (CV 12), "Guanyuan" (CV 4), "Pishu" (BL 20) and "Weishu" (BL 21), etc. The common symptoms and intestinal propulsive rate were observed. The content of I-xylose in serum was detected by phloroglucinol method. Colorimetry method was used to detected content of ATP in jejunum tissues. RESULTS: Compared with group A, the symptom score in group B was increased significantly (both P < 0.01), while the intestinal propulsive rates, the content of D-xylose in serum and ATP in jejunum tissues were decreased significantly (P < 0.05, P < 0.01). Compared with group B, the symptom score in group C and D was decreased significantly (both P < 0.01), while the intestinal propulsive rates, the content of D-xylose in serum and ATP in jejunum tissues were increased significantly (P < 0.05, P < 0.01). There were no significant difference between group C and D (P > 0.05). CONCLUSION: Moxibustion at "Zusanli" (ST 36) etc. could relieve symptoms of spleen-deficiency, enhance motility and absorption functions of small intestine and improve metabolism of small intestine. The efficacy is equal to administration of Sijunzi decoction.

[Effect of plasma of healthy subjects undergoing moxibustion on ethanol-injured human gastric epithelial GES-1 cells in vitro and the involved mitochondrial apoptosis pathway].

OBJECTIVE: To observe the effect of plasma derived from healthy volunteers undergoing moxibustion (moxibustion plasma) on alchol-injured human gastric epithelial GES-1 cells in vitro, and expression of heat shock protein 70 (HSP 70, cell apoptosis inhibitory protein), apoptosis inducing factor (AIF), Smac (a mitochondrial protein), and Caspase 3 and Caspase 9 (the latter 3 proteins are also involved in cell apoptosis) in order to study its mechanisms underlying protecting gastric mucous membrane. METHODS: Twenty-four healthy volunteer subjects (half men and half women) were randomized into acupoint-moximustion (A-M) [Zhongwan(CV 12), Guanyuan (CV 4) and Zusanli (ST 36)] group and non-acupoint-moxibustion (NA-M, 3 cun right to CV 12 and CV 4.1 cun medial to ST 36 ) group (n = 12/group). Moxibustion was applied to the above-mentioned 3 acupoints and non-acupoints for 30 min, once daily for 10 days. Venous blood of the subjects was collected before and after moxibustion. The cultured GES-1 cells were divided into: control group. ethanol-injury group (model), A-M plasma group (A-M-P, plasma got from volunteers undergoing A-M), and NA-M plasma group (NA-M-P,plasma got from volunteers accepting NA-M). The GES-1 cells of the latter 3 groups were treated with 8% ethanol for duplicating cell injury model. Apoptosis was detected by flowcytometry. Expression of HSP 70, second mitochondria-derived activator of Caspase (Smac) and AIF proteins of GES-1 cells were assayed by western blotting, and the immunoactivity of cysteinyl aspirate-specific proteinase-3 and 9 (Caspase-3, 9) was detected by immunocytochemistry. RESULTS: In comparison with the control group, the apoptosis rate, the expression of HSP 70, Smac and AIF proteins, and the immunoactivity of Caspase-3 and Caspase-9 of the model group were increased significantly (P < 0.01). Compared with the model group, the apoptosis rate of GES-1 cells, the expression of Smac and AIF proteins, and the immunoactivity of Caspase-3 and Caspase-9 in the A-M-P group, the apoptosis rate, the expression of Smac and the immunoactivity of Caspase-3 and Caspase-9 in the NA-M-P group were all down-regulated considerably (P < 0.05, P < 0.01). In comparison with the model group, HSP 70 expression of the A-M-P group was up-regulated significantly (P < 0.01). The apoptosis rate of GES-1 cells, the expression levels of Smac, AIF, Caspase-3 and Caspase-9 were significantly lower in the A-M-P group than in the NA-M-P group (P < 0.05, P < 0.01), while the expression of HSP 70 was apparently higher in the A-M-P group than in the NA-M-P group (P < 0.01). CONCLUSION: Plasma derived from the subjects undergoing moxibustion of Zusanli (ST 36), Zhongwan (CV 12) and Guanyuan (CV 4) can inhibit apoptosis of GES-1 cells in vitro, which is closely related to its effects in up-regulating intracellular HSP 70 expression and down-regulating mitochondrial apoptosis protein expression of AIF. Smac, Caspase-3 and Caspase-9.

[Effects of moxibustion on gastric mucosal EGF and TGF-alpha contents and epidermal growth factor receptor expression in rats with gastric mucosal lesion].

OBJECTIVE: To observe the effects of moxibustion on contents of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) expression in the gastric mucosa tissue in rats with gastric mucosal lesion. METHODS: Thirty-six SD rats were randomly and equally divided into control, model and moxibustion groups. Gastric mucosal lesion model was duplicated by restraint and cool water immersion stress. Pre-moxibustion was applied to "Zusanli" (ST 36) and "Zhongwan" (CV 12), "Pishu" (BL 20) and "Weishu" (BL 21) alternately, once everyday for 8 days before modeling. The contents of EGF and TGF-alpha in gastric mucosa were detected by enzyme-linked immunosorbent assay (ELISA) and the expression of EGFR determined by immunohistochemistry. RESULTS: Compared with the control group, only TGF-alpha content in the gastric mucosa in the model group was increased significantly (P < 0.05). Compared with the model group, the EGF and TGF-alpha contents and EGFR immunoactivity in the gastric mucosa were increased significantly in the moxibustion group (P < 0.05, P < 0.01). CONCLUSION: Pre-moxibustion at ST 36, CV 12, BL 20 and BL 21 can up-regulate gastric mucosal EGF and TGF-alpha contents and EGFR protein expression in gastric mucosa lesion rats, which may contribute to its effect in relieving stress-induced gastric mucosal injury.

[Influence of electroacupuncture on myocardial NO and NOS and intracellular Ca2+ contents in myocardial ischemia-reperfusion injury rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on contents of myocardial nitric oxide (NO), nitric oxide synthase (NOS), and intracellular Ca2+ levels in experimental myocardial ischemia-reperfusion injury (MI/RI) rats, so as to provide some evidence for acupuncture treatment of cardiovascular disorders and for the correlation between the acupoints and the internal organs. METHODS: Fifty male Wistar rats were randomly divided into sham-operation (sham), model, Neiguan (PC 6), Lieque (LU 7), and Hegu (LI 4) groups (n = 10/group). MI/RI model was established by occlusion of the descending anterior branch of the left coronary artery for 40 min and reperfusion for 60 min. EA (30 Hz/100 Hz, 2-4 mA) was applied to the bilateral PC 6, LU 7 and LI 4 for 20 min. The myocardial tissue of the ischemic area was taken, homogenated and centrifuged, separated for assaying NO and NOS activity by using spectrophotometer and their contents with nitrate reductase colorimetric assay. Another part of the myocardium tissue was processed to be cell suspension (added with Fluo-3/AM dye) for assaying intracellular free [Ca2+]i concentration under the laser confocal microscope. RESULTS: Compared with sham group, myocardial NO and NOS contents of model group decreased mildly and significantly (P < 0.05), while myocardial fluorescence intensity (content) of intracellular Ca2+ increased evidently (P < 0.01). In comparison with model group, the contents of myocardial NO and NOS were upregulated considerably in PC 6 group (P < 0.05), and intracellular Ca2+ content in PC 6 group reduced significantly (P < 0.01). No significant differences were found between LU 7 and model groups and between LI 4 and model groups in the above mentioned 3 indexes (P > 0.05). CONCLUSION: EA of PC 6 can upregulate myocardial NO and NOS contents and downregulate myocardial intracellular Ca2+ level in MI/RI rats, which may contribute to its effect in relieving myocardial injury. Neiguan (PC 6) and the heart have a relatively specific correlation.

[Effect of different frequency electroacupuncture on the expression of substance P and beta-endorphin in the hypothalamus in rats with gastric distension-induced pain].

OBJECTIVE: To observe the effect of different frequency electroacupuncture (EA) on hippocampal substance P (SP) and beta-endorphin (beta-EP) in rats with gastralgia so as to explore its underlying mechanism in preventing and relieving visceral pain. METHOD: Fifty rats were randomly divided into control, sham operation, model, low- frequency-EA and high frequency-EA groups. Gastralgia model was duplicated by gastric distension (a pressure of 40 mmHg) with a latex balloon. Low (2 Hz) and high (100 Hz) frequency EA was applied to bilateral "Zusanli (ST 36)" for 30 min. Pain score was given by using Al-Chaer's method (abdominal withdrawal reflex) and the expression of hypothalamic beta-EP and SP was detected by immunohistochemistry. RESULTS: Both low- and high-frequency EA of ST 36 could significantly lower the pain score (P < 0.01), and the effect of high-frequency EA was markedly superior to that of low-frequency (P < 0.01). Compared with control and sham-operation groups, both SP immuno-reaction (IR) and beta-EP IR positive cells in the hypothalamus in model group increased significantly in the number (P < 0.01), suggesting an upregulation of SP and beta-EP expression after occurrence of gastralgia. In comparison with model group, the number of SP IR and (beta-EP IR positive cells in the hypothalamus in low frequency and high frequency EA groups increased further (P < 0.01) particularly in low frequency EA group. CONCLUSION: Both low-frequency and high-frequency EA of "Zusanli (ST 36)" can effectively relieve gastric distension-induced gastralgia in the rat, and the effect of high-frequency EA is better, which may be closely related to its actions in promoting the release and synthesis of hypothalamic beta-EP and SP.

[Optimization of extraction procedure of psammosilene tunicoides by uniform design with pharmacodynamic index].

OBJECTIVE: For anti-inflammation, relieving pain and decreasing acute toxicity, the best condition for extracting procedure of Psammosilene tunicoides was chosen. And the relationships between several solvent extracts and pharmacodynamic index were studied. METHOD: Uniform design with multi-targets was used in the optimization process, and the evaluate index were the results of anti-inflammation, relieving pain and LD50 were employed as the evaluating indexes. At the same time, the contents of the extracts, water extract, acetic ester extract and ether extract were determined. RESULT: The best condition is that the medicinal material is optimized with 8 times of water (pH 6-7) and 2 h of each time. The best condition is satisfactory by test verification. And the pharmacodynamic actions of anti-inflammation, relieving pain and LD, were not obviously related to the dry extracts, water extract, acetic ester extract and ether extract. CONCLUSION: Uniform design method with pharmacodynamic index has the merit of being relaated to the function and symptom directly. The extracted twice technology condition optimized is stable, feasible and suitable for industry production. And it is not suitable to take the solvent extracts as quality control index related to the function and symptom.

[Effect of electroacupuncture of "Neiguan" (PC 6) on Na+-K+-ATPase activity and its gene expression in cardiocyte membrane in rats with myocardial ischemia-reperfusion injury].

OBJECTIVE: To compare the effects of electroacupuncture (EA) of "Neiguan" and other acupoints on Na+ -K+ -ATPase activity and its gene expression in ischemic myocrardial cell membrane in rats. METHODS: A total of 50 Wistar rats were randomly divided into pseudo-operation (control), model, Neiguan (PC6), Shenmen (HT7) and Hegu (LI4) groups, with 10 rats in each group. Myocardial ischemia-reperfusion (I/R) model was duplicated by occlusion of the descending anterior branch of the left coronary artery and reperfusion. EA (30/100 Hz, 2-4 mA) was applied to the above-mentioned acupoints for 20 min respectively. Na+ -K+ -ATPase activity was determined by inorganic phosphorus colorimetry, and Na+ -K+ -ATPase gene expression was detected using reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: Compared with control group, the activity of Na+ -K+ -ATPase in model, PC6, HT7 and LI4 groups decreased significantly after myocardial I/R (P<0.05, 0.01); while in comparison with model, the activity of Na+ -K+ -ATPase in PC6 group increased considerably (P<0.01). Regarding the expression of Na+ -K+ -ATPase mRNA, compared with control group, it was down-regulated significantly in model, HT7 and LI4 groups (P<0.01); while in PC6 group, Na+ -K+ -ATPase mRNA expression was up-regulated markedly compared with model group (P<0.05); The activity of Na+ -K+ -ATPase and the expression of Na+ -K+ -ATPase mRNA in HT7 and LI4 groups were significantly lower than those in PC6 group (P<0.01, 0.05). No significant differences were found between HT7 and LI4 groups in the activity of Na+ -K+ -ATPase and the expression of Na+ -K+ -ATPase mRNA (P>0.05). CONCLUSION: EA of "Neiguan" (PC6) may potentiate the activity of Na+ -K+ -ATPase and up-regulate Na+ -K+ -ATPase mRNA expression, which may contribute to its protective effect on ischemic cardiocytes.

[Effects of pricking bleeding at "Weizhong" (BL40) on sciatic nerve conduction velocity and interleukin-1alpha content in the lumbar vertebral nucleus pulposus in experimental lumbar intervertebral disc protrusion rabbits].

OBJECTIVE: To observe the effect of "Weizhong" (BL40) bleeding on sciatic nerve conduction velocity (SNCV) and interleukin-1alpha (IL-1alpha) content of the lumbar vertebral nucleus pulposus tissue in experimental lumbar intervertebral disc protrusion (LIDP) rabbits so as to explore the underlying mechanism of blood letting in the treatment of LIDP. METHODS: Forty healthy New Zealand rabbits were randomly assigned to control, model, BL40 and non-point (1 cm medial to BL40) groups, with 10 cases in each group. LIDP model was established by pushing the intervertebral disc tissue rightward to protrude the posterior longitudinal ligament with a self-made instrument after exposing the L6-7 vertebrae and intervertebral space. Blood-letting was performed at "Weizhong" (BL40) and non-point by using a triangle-edged needle to prick the local tissue, once daily and continuously for 7 days. SNCV was detected by using BL-410 physiological signal recording system, and the content of IL-1alpha in L6-7 intervertebral disc tissue determined with enzyme linked immunsorbent assay (ELISA). RESULTS: In comparison with control group and pre-modeling, SNCV decreased significantly in model, BL40 and non-acupoint groups after LIDP; while compared with model group, values of SNCV in BL40 group increased considerably (P<0.05), no significant changes were found between non-acupoint group and model group (P>0.05). Compared with control group, the contents of IL-1alpha in the nucleus pulposus in model and non-acupoint groups increased remarkably (P<0.05, 0.01), but no significant difference was found between control and BL40 groups in IL-1alpha level (P>0.05). CONCLUSION: Blood-letting at "Weizhong" (BL40) can relieve LIDP-induced nerve injury and mechanical oppression to improve SNCV, which may be closely related to its effect in decreasing inflammatory factor IL-1alpha level.

[Preparation and quality control of soybean isoflavone dropping pills].

OBJECTIVE: To establish the optimized preparation procedure and study the method to determine the content for soybean isoflavone(SIF) Dropping Pills. METHOD: The preparation conditions, such as the proportion between SIF and PEGs, the temperature of mixture of SIF and PEGs, dropping distance, etc., were studied with Uniform Design and One-way ANOVA. SIF was identified by TLC and the content of SIF was determined by UV spectrometry at 262 nm detection wavelength. RESULT: Three batches of the prepared products meet the standards of the Chinese pharmacopoeia on dropping pills. SIF can be identified by TLC. Using UV spectrometry, the linear range of SIF was 0. 407 2 to 4. 072 g x mL(-1) and the correlation coefficient was 0. 999 8. In high, middle and low concentration, average recovery were 96. 54%, 97.27% and 97.21%, respectively (RSD were 1.3%, 0.78% and 0.71%). CONCLUSION: The preparation procedure is feasible, simple and suitable, the method established in this paper can be adopted for the quality control of SIF dropping pills, and the determination method is simple, relatively fast and accurate.

[Expressions of epidermal growth factor receptor signaling substances in gastric mucosal cells influenced by serum derived from rats treated with electroacupuncture at stomach meridian acupoints].

OBJECTIVE: To study the effects of serum derived from rats treated with electroacupuncture at stomach meridian acupoints on the expressions of epidermal growth factor receptor (EGFR) signaling substances phospholipase C gamma-1 (PLC gamma-1), protein kinase C (PKC) and c-myc in gastric mucosal cells. METHODS: Sixty rats were randomly divided into normal group, stomach meridian group, gallbladder meridian group, stomach meridian plus PD153035 group and gallbladder meridian plus PD153035 group. Water-immersion and restrained stress methods were adopted for inducing gastric mucosal injury in the rats. Gastric mucosal cells were separated by using pronase digestion method, and incubated by PD153035, a EGFR inhibitor, and 100 ml/L serum. The expression of PLC gamma-1 in the gastric mucosal cells was tested by enzyme linked-immunosorbent assay (ELISA), while the expression of PKC by isotope incorporate assay and the expression of c-myc by reverse transcription polymerase chain reaction assay (RT-PCR). RESULTS: In gastric mucosal cells, weak expressions of PLC gamma-1, PKC and c-myc were seen in the normal group, and relatively strong expressions of PLC gamma-1, PKC and c-myc were seen in the stomach meridian group and the gallbladder meridian group, among which, the expressions of PLC gamma-1, PKC and c-myc in the stomach meridian group were the strongest, and there was a significant difference between the stomach meridian group and the gallbladder meridian group (P<0.01). Relative weak expressions of PLC gamma-1, PKC and c-myc were seen in the stomach meridian plus PD153035 group and the gallbladder meridian plus PD153035 group, and there was a significant difference between the stomach meridian group and the stomach meridian plus PD153035 group (P<0.01). CONCLUSIONS: The serum derived from the rats treated with electroacupuncture at stomach meridian acupoints can activate the EGFR singling pathway, and this provides an evidence for the theory of "relative particularity between meridians and viscera" in traditional Chinese medicine.