Effect of electroacupuncture on calcium-activated chloride channel currents in interstitial cells of Cajal in rats with diabetic gastroparesis / 针灸推拿医学（英文版）

Objective: To investigate the mechanisms of electroacupuncture (EA) at Zusanli (ST 36), Liangmen (ST 21) and Sanyinjiao (SP 6) in intervening diabetic gastroparesis (DGP) based on calcium-activated chloride channel. Methods: Forty Sprague-Dawley rats were randomly divided into four groups, including a normal control group (group A), a model group (group B), an EA group (group C) and a metoclopramide group (group D), with 10 rats in each group. A single intraperitoneal injection of 2％ streptozotocin (STZ) combined with 8-week high-glucose high-fat diet was used to establish a DGP rat model. After intervention, gastrointestinal propulsive rate was observed; the expression level of transmembrane protein 16A (TMEM16A) was examined by immunohistochemistry; the Ca2+ concentration in interstitial cells of Cajal (ICCs) was detected by immunofluorescence; and whole-cell patch-clamp technique was applied to detect the current intensity of calcium-activated chloride channel (ICaCC) in ICCs in gastric antrum. Results: After modeling, the blood glucose levels in group B, group C and group D were significantly increased compared with group A (all P<0.01); after intervention, compared with group B, the blood glucose levels in group C and group D were significantly decreased (P<0.05, P<0.01); the intra-group comparison of blood glucose level between after modeling and after intervention found significant difference only in group C (P<0.01). The gastrointestinal propulsive rates in group B, group C and group D were significantly different from that in group A (P<0.01 or P<0.05); the gastrointestinal propulsive rates were markedly higher in group C and group D than in group B (P<0.01, P<0.01). The expressions of TMEM16A in group B and group C were decreased compared with group A (P<0.01, P<0.05); the expressions of TMEM16A in group C and group D were increased compared with group B (P<0.01, P<0.05). The fluorescence intensity of Ca2+ was significantly lower in group B than in group A (P<0.01); the fluorescence intensity of Ca2+ was significantly higher in group C and group D than in group B (P<0.01, P<0.05). ICaCC in ICCs in group B was significantly decreased compared with group A; ICaCC in group C and group D were increased compared with group B. Conclusion: EA at Zusanli (ST 36), Liangmen (ST 21) and Sanyinjiao (SP 6) can significantly improve gastrointestinal motility in DGP rats by up-regulating the ICaCC in ICCs.

Establishment of a cell model of insulin-resistant 3T3-L1 adipocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the optimal conditions for establishing insulin-resistant 3T3-L1 adipocytes.</p><p><b>METHOS</b>Dexamethason (DEX), 3-isobutyl-methylxanthine (IBMX) and different concentrations of insulin (10(-8), 10(-7), and 10(-6) mol·L(-1)) were used to induce 3T3-L1 preadipocytes into mature adipocytes identified by oil red O staining. We established insulin- resistant 3T3-L1 adipocytes cell model (IR-3T3-L1) by exposing the cells to 1µmol·L(-1) DEX, and the changes of glucose concen- tration in the cell culture were determined by glucose oxidase-peroxidase (GOD-POD) assay.</p><p><b>RESULTS</b>Treatment of 3T3-L1 cells with DEX, IBMX and 10(-6) mol·L(-1)) insulin for 9 days resulted in the differentiation of >90% of the cells into mature adipocytes. IR-3T3-L1 cells cultured for 96 h in the culture media containing 1 µmol·L(-1) DEX showed significantly increased glucose consumption (P=0.0003) as compared with the control group at 36 h (P<0.001).</p><p><b>CONCLUSION</b>3T3-L1 cells can be induced into mature adipocytes by exposure to 1 µmol·L(-1) DEX, 0.5 mmol·L(-1) IBMX and 10(-6) mol·L(-1)) insulin. A 96 h exposure to 1 µmol·L(-1) DEX can induce 3T3-L1 adipocytes to acquire insulin resistance that can be maintained for 36 h.</p>

Effects of rutaecarpine on inflammatory cytokines in insulin resistant primary skeletal muscle cells / 中国中药杂志

It is now well established that inflammation plays an important role in the development of numerous chronic metabolic diseases including insulin resistance (IR) and type 2 diabetes (T2DM). Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Our pre- vious study has shown that rutaecarpine (Rut) can benefit blood lipid profile, mitigate inflammation, and improve kidney, liver, pan- creas pathology status of T2DM rats. However, the effects of Rut on inflammatory cytokines in the development of IR-skeletal muscle cells have not been studied. Thus, our objective was to investigate effects of Rut on inflammatory cytokines interleukiri (IL)-1, IL-6 and tumor necrosis factor (TNF)-α in insulin resistant primary skeletal muscle cells (IR-PSMC). Primary cultures of skeletal muscle cells were prepared from 5 neonate SD rats, and the primary rat skeletal muscle cells were identified by cell morphology, effect of ru- taecarpine on cell proliferation by MTT assay. IR-PSMC cells were induced by palmitic acid (PA), the glucose concentration was measured by glucose oxidase and peroxidase (GOD-POD) method. The effects of Rut on inflammatory cytokines IL-1, IL-6 and TNF-α in IR-PSMC cells were tested by enzyme-linked immunosorbent assay (ELISA) kit. The results show that the primary skeletal muscle cells from neonatal rat cultured for 2-4 days, parallel alignment regularly, and cultured for 7 days, cells fused and myotube formed. It was shown that Rut in concentration 0-180. 0 μmol x L(-1) possessed no cytotoxic effect towards cultured primary skeletal muscle cells. However, after 24 h exposure to 0.6 mmol x L(-1) PA, primary skeletal muscle cells were able to induce a state of insulin resistance. The results obtained indicated significant decrease (P < 0.05 to P < 0.001) IL-1, IL-6 and TNF-α production by cultured IR-PSMC cells when incubating 24 hours with Rut, beginning from 20 to 180.0 μmol x L(-1). IL-1, IL-6 and TNF-α in the Rut treated groups were dose-dependently decreased compared with that in the IR-PSMC control group. Our results demonstrated that the Rut promoted glucose consumption and improved insulin resistance possibly through suppression of inflammatory cytokines in the IR-PSMC cells.

SSR information in Erigeron breviscapus transcriptome and polymorphism analysis / 中国中药杂志

<p><b>OBJECTIVE</b>The SSR information in the transcriptome of Erigeron breviscapus was analyzed in this study, in order to further develop new functional genes SSR markers laid a solid foundation.</p><p><b>METHOD</b>SSR loci were searched in all of 52,060 unigenes by using est_timmer. Perl program and SSR primers were designed by Primer3. Furthermore, 36 pairs of primers were randomly selected for the polymorphism analysis on 13 Erigeron breviscapus plants collected from different places.</p><p><b>RESULT</b>A total of 3639 SSRs were found in the transcriptome of Erigeron breviscapus, distributed in 3260 unigenes with the distribution frequency of 6.99%. Di-nucleotide repeat was the main type, account for as much as 34.41% of all SSRs, followed by mono-nucleotide (31.41%) and tri-nucleotide repeat motif (28.08%). The di-nucleotide repeat motifs of AT/AT and AC/GT were the predominant repeat types (28.71%). The tri-nucleotide repeat motifs of AAT/AT was the predominant repeat types (7.94%). For validation the availability of those SSR primers, we randomly selected 36 pairs of primers for PCR amplification. Among them, 34 pair primers (94.44%) produced clear and reproductive bands, 19 pair primers showed polymorphism (52.78%), and 13 Erigeron breviscapus plants were divided into 2 groups.</p><p><b>CONCLUSION</b>There are numerous SSRs in Erigeron breviscapus transcriptome with high frequency and various types, this will provide abundant candidate molecular markers for genetic diversity study and genetic map in this plant.</p>

Genetic diversity and group consistency of breeding strains of Erigeron breviscapus determined by AFLP marker / 中国中药杂志

<p><b>OBJECTIVE</b>To analyze the genetic diversity and breeding strains of the E. breviscapus germplasms, in order to provide theoretical information for Erigeron breviscapus breeding.</p><p><b>METHOD</b>The genetic diversity and genetic structure were assayed to six germplasm resource of E. breviscapus which collected from Yunnna with 11 pairs primers and AFLP molecular marker.</p><p><b>RESULT</b>Six hundred and four amplification bands among 636 DNA bands were from six accession of E. breviscapus, which are about 82.40% of total bands. The six germplasms could be divided into three group at the 0. 706 similarity coefficient level. The first category include QS-1, QS-2 and Dali, Shilin, Kunming population. The second category included wild population of Qiubei. The third category included several sample from different district. The mean genetic similarity coefficient of QS-1 and QS-2 was bigger, genetic similarity coefficient range was smaller, hereditary character was more stable. Molecular system clustering analysis showed that the geographical origin of the same part had relative polymerization phenomenon and its genetic relationship was close. Qiubei was a single group possibly relating to the specific genetic basis.</p><p><b>CONCLUSION</b>The analysis of genetic diversity of E. breviscapus by AFLP marker is reliable. The systematic E. breviscapus breeding is feasible.</p>

Simultaneous determination of chlorogenic acid, scutellarin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid in different parts of Erigeron breviscapus by high-performance liquid chromatography / 中国中药杂志

<p><b>OBJECTIVE</b>A high-performance liquid chromatographic (HPLC) method was developed for simultaneous determination of chlorogenic acid, scutellarin, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid in different parts of Erigerontis Herba.</p><p><b>METHOD</b>The four constituents were measured on an Agilent Zorbax SB-C18 column (4.6 mm x 450 mm, 5 microm) with a gradient elution of acetonitrile (A) -0.3% phosphoric acid solution (B) (0-10 min, 12%-15% A, 10-32 min, 15% A, 32-33 min, 15%-20% A, 33-50 min, 20%-22% A) at wavelength of 335 nm and 327 nm, and a flow rate of 1.0 mL x min(-1) and the column temperature was 30 degrees C.</p><p><b>RESULT</b>Linearity of each standard was established in the concentration range of 0.050 1-1.002 microg for chlorogenic acid, 0.165 9-3.318 microg for chlorogenic acid, 0.049 7-0.994 microg for 3,5-dicaffeoylquinic acid, 0.048 7-0.974 p.g for 4,5-dicaffeoylquinic acid respectively, with correlation coefficient r > 0.999 6. Average recoveries (n = 6) of 4 compounds were 98.53% with a RSD of 0.94%, 99.68% with a RSD of 0.49%, 98.78% with a RSD of 1.1%, 99.06% with a RSD of 0.81%, respectively.</p><p><b>CONCLUSION</b>The developed method is simple, accurate, and precise, it can be used for the quantitative analysis of Erigeron breviscapus.</p>

Cloning and bioinformation analysis of flavone synthase II gene of Erigeron breviscapus / 中国中药杂志

<p><b>OBJECTIVE</b>Erigeron breviscapus is a medicinal plant with the most developmental potential in Yunnan province, which is belongs to Erigeron genus of Compositae family. Scutellarin, the main active component of Erigeron breviscapus is one of flavone 7-O-glucuronide derivatives, its biosynthesis pathway is still not clear.</p><p><b>METHOD</b>Full length cDNA encoding flavone syhthase II gene in E. breviscapus was cloned in this study using R-PCR, 3'-RACE and 5'-RACE.</p><p><b>RESULT</b>The opening reading frame of FS II cDNA of E. breviscapus is 1 557 bp long and encoding 518 amino acids, designed as EbFS II, which is highly homologous with FS II of Compositae species, like Callistephus chinensis, Cynara cardunculus var. scolymus, Gerbera hybrida, Dahlia pinnata and Lobelia erinus.</p><p><b>CONCLUSION</b>Phylogenetic analysis showed that EbFS II might has the function of directly converting flavanone to flavone.</p>