Correlations between content of linarin in Chrysanthemum indicum and climatic factors in habitats / 中国中药杂志

Chrysanthemi Indici Flos(CIF), the capitulum of Chrysanthemum indicum, is widely used in proprietary Chinese medicine and daily chemical products. At present, CIF is mainly produced from wild resources and rarely cultivated. This study aims to reveal the correlations between linarin content in CIF and climatic factors in different habitats, and provide a theoretical basis for suitable zoning and rational production of medicinal materials. The content of linarin in CIF was determined by HPLC. Grey relational analysis and Pearson correlation analysis were carried out for linarin content with climatic factors. The results showed that the content of linarin in CIF was significantly different among different habitats. The grey relational degrees of climatic factors with linarin content was in an order of average annual precipitation>annual average sunshine hours>annual average temperature>longitude>annual frost-free period>latitude>altitude. Longitude, annual average temperature and average annual precipitation had significantly positive correlations with the content of linarin in CIF, whereas latitude and altitude showed negative correlations with it. The annual frost-free period and annual average sunshine hours had no significant correlation with the content of linarin in CIF. The content of linarin in CIF varied significantly in different habitats. High longitude, low latitude, low altitude, high annual average temperature and high annual average precipitation could be used as indicators for the habitats of high-quality Ch. indicum. This study provides a reference for selecting suitable producing areas of Ch. indicum and establishing artificial cultivation system.

Study on wild tending of Chrysanthemum indicum / 中国中药杂志

To provide theoretical basis for the formation of Chrysanthemum indicum resource and quality,the differences in biology,ecology,yield and quality of Ch. indicum population in the wild tending,the cultivated and the wild were analyzed and compared in this study. The results showed that in the aspect of biology,there was no significant difference between the condition of wild tending and cultivated Ch. indicum at the height of plant,both were significantly higher than wild Ch. indicum. In the aspect of ecology,in the condition of wild tending is less likely to occur water logging than cultivated Ch. indicum. In the condition of wild tending is less likely to occur plant diseases and insect pests than Ch. indicum in the condition of wild. In the aspect of production,the average output of the sample of the condition of wild tending was significantly higher than that the condition of artificial cultivation and wild quadrat. There was no significant difference between the three kinds of condition on the traits of capitulum dichasium,the content of buddleoside of the wild tending Ch. indicum was significantly higher than that of 0. 80% required by the Chinese Pharmacopoeia( 2015 edition). Therefore,the artificial supplement and the appropriate artificial management of wild tending is a mode of production to increase the population density of Ch. indicum and obtain high quality and high yield medicinal materials.

Effect of Chrysanthemum indicum on absorption characteristics of Cd and its effect on quality of medicinal materials / 中国中药杂志

The Cd stress of Chrysanthemum indicum was treated by different concentrations of Cd Cl2 solution in the culture substrate. The content of Cd in different parts of Ch. indicum and the content of buddleoside and the total flavonoids in Ch. indicum were determined. The absorption characteristics of Cd elements in Ch. indicum were analyzed. And the influence of Cd elements on the quality of the herbs. The results showed that the application of soil Cd in the range of 0-100 mg·kg~(-1) had no significant effect on the biomass of Ch. indicum,and the root-shoot ratio showed a decreasing-increasing-decreasing trend. The content of Cd in different parts of Ch. indicum was significantly different,and the content of aboveground part was higher than that of underground part. The enrichment factors of Cd elements in different parts of Ch. indicum are different. The enrichment coefficient of aboveground parts is larger than that of underground parts. The whole parts and plants show an increase first and then decrease,and the overall enrichment factor is greater than1. The transfer coefficient of the aerial part/underground part of Ch. indicum showed a decreasing-increasing-decreasing-increasing trend with the increase of the amount of Cd applied in the soil,and the transfer coefficient was higher than 1. The contents of buddleoside and total flavonoids in Ch. indicum after Cd stress treatment were lower than the control,and the overall performance was lower and then increased,but it was still significantly lower than the control,indicating that Cd pollution directly led to the decrease of chemical quality of Ch. indicum.

Identification of Chrysanthemum indicum in different geographical populations and Ch. morifolium based on DNA barcodes of psbA-trnH,matK and trnL / 中国中药杂志

DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum and Ch. morifolium based on psbA-trn H,mat K and trn L sequences. The total DNA was extracted from 21 samples collected,and the psbA-trn H,mat K,trn L sequences were amplified by PCR and sequenced. The information of these sequences were obtained. We aligned all 63 sequences,calculated the intraspecific and interspecific distances,analysed the SNPs distribution of psbA-trn H+mat K+trn L combination sequences and constructed the Neighbor-joining( NJ) Tree,using MEGA 7. 0. The results showed that the genetic distances of Ch. indicum,Ch. indicum( Juhuanao)and Ch. morifolium were overlapped. The SNPs analysis of psbA-trn H+mat K+trn L combination sequences showed that there were 19 nucleotide polymorphism loci( SNPs) and nine parsim-informative sites in the combination sequences. In addition,Ch. indicum showed more obvious sequence polymorphism than those of Ch. indicum( Juhuanao) and Ch. morifolium. The psbA-trn H sequences showed obvious length variation.The NJ Tree showed that Ch. morifolium numbered C2-C5 were clustered into a single subbranch with a bootstrap value of 62%,and Ch.morifolium could be distinguished from Ch. indicum and Ch. indicum( Juhuanao). Moreover,Ch. indicum numbered Z9 and Z10 collected from Gansu province were singly clustered into one branch with a bootstrap value of 77%. It was also found that the changes of psbA-trn H and trn L sequences information of Ch. indicum samples from the northwest were obviously related to the geography and environment. Moreover,Ch.indicum and Ch. indicum( Juhuanao) had obvious differentiation,were also regarded as the evolutionary sources of Ch. morifolium. Therefore,psbA-trn H+mat K+trn L combination sequences as DNA barcode can identify Ch. indicum and Ch. morifolium accurately and rapidly,which provides an important basis for germplasm resources identification and species identification.

Comparative study on content of heavy metals in Chrysanthemum indicum and soil in different areas / 中国中药杂志

ICP-MS was used to determine the contents of five heavy metals such as lead (Pb), cadmium (Cd), mercury (Hg), arsenic (As) and copper (Cu) in Chrysanthemum indicum and soil samples. Pearson correlation and path analysis were used to analyze the effects of latitude and longitude and altitude on the content of heavy metal elements in Ch. indicum. To explore the relationship between geographical location of heavy metal content in Ch. indicum and the content of heavy metal elements in soil. The results showed that the Ch. indicum had high enrichment of Cd and had a synergistic effect on the absorption of five heavy metal elements. Elevation directly affected the accumulation of Pb and Cd in Ch. indicum and was a positive effect. The direct positive effect of latitude acts on the accumulation of Hg and Cu in Ch. indicum. The effect of longitude on the Cd content was the negative effect of longitude. The geographical location had different effects on the accumulation of heavy metal elements in Ch. indicum. There was a certain interaction between direct and indirect effects. Ch. indicum had different absorption and enrichment characteristics for different heavy metal species. When controlling the heavy metal content and medicinal material quality of Ch. indicum, we must fully consider the influence of geographical location on the quality of medicinal materials, select suitable geographic regions to carry out acquisitions, and choose a good ecological environment. Regional establishment of Chinese herbal medicine planting bases, or adopting certain agricultural measures to reduce the absorption of heavy metal elements in Ch. indicum herbs, while conducting research on chemical composition, strengthening the research on the effective forms of heavy metals, and paying attention to the safety of medicinal herbs.

Cadmium Activates Reactive Oxygen Species-dependent AKT/mTOR and Mitochondrial Apoptotic Pathways in Neuronal Cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells.</p><p><b>METHODS</b>Neuronal cells (primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl-L-cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays.</p><p><b>RESULTS</b>Cd-induced activation of Akt/mTOR signaling, including Akt, mTOR, p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G).</p><p><b>CONCLUSION</b>Cd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.</p>

Epidemiological survey of childhood asthma in 2010 in urban Baotou, China / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the prevalence rate of childhood asthma in 2010 in urban Baotou, China, as well as the characteristics of attacks and the status of diagnosis and treatment of childhood asthma.</p><p><b>METHODS</b>More than 10 000 children (0-14 years) were selected from 3 secondary schools, 3 primary schools, 6 kindergartens, and 4 community vaccination sites in urban Baotou by cluster random sampling between September 2009 and August 2010. A standardized preliminary questionnaire was used for screening out suspected cases, which were then confirmed or excluded by a clinician; the confirmed cases underwent further questionnaire survey. Double entry and validation was adopted for all data using Epi-Info software, and analysis was performed using SPSS 13.0.</p><p><b>RESULTS</b>A total of 11 323 children were surveyed. Asthma was diagnosed in 127 cases (including 121 children with typical asthma and 6 children with cough variant asthma), with a prevalence rate of 1.12%. The prevalence rate of asthma in male children was significantly higher than that in female children (1.51% vs 0.72%; P<0.01). The prevalence rate of asthma in 2010 was significantly increased compared with that in 1990 (0.55%) and 2000 (0.88%) (P<0.05). Systemic glucocorticoid use decreased significantly from 60.2% in 2000 to 25.9% in 2010 (P<0.01); inhaled corticosteroid use increased significantly from 13.6% in 2000 to 85.8% in 2010 (P<0.01); antibiotic use decreased from 98.1% in 2000 to 66.9% in 2010 (P<0.01). The multivariate logistic regression analysis showed that family history of allergy, allergic rhinitis, chronic cough, and recurrent respiratory tract infection were independent risk factors for childhood asthma.</p><p><b>CONCLUSIONS</b>The prevalence rate of childhood asthma in urban Baotou shows an increasing trend. Inhaled corticosteroids have been widely used.</p>

Establishment of human cardiac C protein induced experimental autoimmune myocarditis model in rat / 中华心血管病杂志

<p><b>OBJECTIVE</b>To construct the recombinant plasmid of human cardiac C protein (CCP) peptide with immunogenicity and to express, purification and renature fusion protein. The fusion protein was injected to Lewis rats to establish experimental autoimmune myocarditis (EAM) model.</p><p><b>METHODS</b>Total RNA was extracted from human heart and used as the template for reverse transcriptase-directed cDNA synthesis. The cDNA was then amplified by polymerase chain reaction (PCR) using oligonucleotide primers specific for CCP peptide with immunogenicity. Subsequently, the purified CCP peptide gene was cloned into PEASY-T1 vector and the ligated product was identified by PCR and DNA sequence analysis. Then the CCP target gene of positive clone was inserted into the pQE30, a prokaryotic expression vector, and the inserting plasmid was transformed into Escherichia coli. host M15. The positive clone extracted from the bacterium liquid was sieved by insertional inactivation sieve method and identified by PCR of bacterium liquid, CCP immunological peptide was purified and renatured in semipermeable membrane. EAM model in Lewis rats was induced by injection of mixture of 100 µg CCP fusion protein immunological peptide and 2.5 g/L completed Freund adjuvant from two double foot pad and subsequent abdominal injection of 0.5 µg pertussis toxin. Two, four, six, and eight weeks after immunization, hemodynamic evaluation was made and hearts underwent histological examination.</p><p><b>RESULTS</b>The DNA sequence analysis for cloning vector extraction revealed that the CCP target gene was cloned into pQE30 exactly. The DNA of 1000 bp length was obtained by PCR examination of bacterium liquid with transformation of express recombinants which were consistent with the expected size. Purified fusion protein in vertical slab gel electrophoresis showed 35 000 as expected. The recombinant CCP fusion protein existed in inclusion bodies of E. coli and amounted to 80% - 90% of the total protein. Hemodynamic and histological evaluations showed typical acute inflammatory responses at 2 weeks, subacute inflammatory and fibrosis changes at 4 weeks after injection, and signs of chronic dilated cardiomyopathy at 6 weeks post injection.</p><p><b>CONCLUSION</b>Combination of gene clone technique and histidine tag protein purification technique can be used to synthesize human cardiac C protein to induce EAM model in Lewis rat.</p>

Experimental study of MMP-2 inhibitor treatment of experimental autoimmune myocarditis in Lewis rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitor of matrix metalloproteinase-2 (MMP-2) (2R)-2-[5-[4-[ ethyl-methylamino] phenyl [thiophene-2-sulfonylamino]-3-methylbutyric acid (TISAM) therapeutic effect on experimental autoimmune myocarditis (EAM) in Lewis rats.</p><p><b>METHODS</b>Treatment protocol of oral administration of 5 mg/kg TISAM once a day for 14 days was performed on EAM Lewis rats. EAM Lewis rats were divided into 3 groups: treatment in early, middle and later stage respectively (n = 20). After experiment at the designate time point, the rats were euthanatized and hearts were harvested. Cardiac inflammatory score, fibrosis score and content, and infiltration of macrophages and T lyminflammatory score, fibrosis score and content, and infiltration of macrophages and T lymphocytes, message RNA (mRNA) expression of matrix metalloproteinase (MMP)-2 and MMP-9 and protein activity of gelatinase were determined.</p><p><b>RESULTS</b>TISAM treatment in early phase was invalid (treatment started from the creation of the model), treatment in middle and later phase was effective (treatment started from 7 and 14 day after the creation of the model).</p><p><b>CONCLUSION</b>Inhibitor of MMP-2 can block ventricular remodeling in middle stage in EAM Lewis rats. The mechanism maybe alleviate the inflammatory cell cardiac infiltration, decrease the mRNA expression of MMP-2 at transcript level and downregulate gelatinase activity at protein level.</p>

Effects of matrix metalloproteinase-9 inhibitor in Lewis rats with experimental autoimmune myocarditis / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effects of matrix metalloproteinase-9 (MMP-9) inhibitor minocyclin hydrochloride in Lewis rats with experimental autoimmune myocarditis (EAM).</p><p><b>METHODS</b>EAM was induced by injection of cardiac C protein emulsified in completed Freund adjuvant in double footpad and intraperitoneal injection of pertussis toxin on 6- to 8-week old Lewis rats. Sixty EAM Lewis rats were divided into 3 groups (early, middle and late intervention groups, n = 20 each: 10 minocyclin treated and 10 control rats). In early intervention group, rats in treatment group received intraperitoneal injection of minocyclin hydrochloride from 1(st) to 21(st) day after immunization; in middle intervention group, rats were treated from 8(th) to 28(th) day after immunization and in late intervention group, rats were treated from 15(th) to 35(th) day after immunization (50 mg/kg body weight, once daily). Control rats received intraperitoneal injection of same volumetric physiological saline at corresponding time periods. At the end of intervention, rats were euthanatized and hearts were harvested. Paraffin sections were used for hematoxylin and eosin stain to determine the inflammatory score, for picrosirius stain to determine fibrosis score and collagen content, and for immunohistological stain to determine macrophages and T lymphocytes. Real time PCR was used to detect mRNA expression of myocardial MMP-2 and MMP-9. Cryostat sections were used for in situ zymography to detect protein activity of gelatinase.</p><p><b>RESULTS</b>Inflammatory score in cardiac paraffin slides, number of cardiac macrophages and T lymphocytes, cardiac interstitial fibrosis score and content, expression of MMP-2, 9 mRNA and activity of gelatinase in treatment group were all significantly lower than in control group for early and middle intervention groups (inflammatory score: early control group vs. treatment group: 3.03 ± 1.35 vs.1.51 ± 0.36, P < 0.05, middle control group vs. treatment group: 3.75 ± 0.29 vs. 2.11 ± 0.82, P < 0.01; cardiac interstitial fibrosis score, early control group vs. treatment group: 2.75 ± 0.29 vs.1.51 ± 0.35, P < 0.01, middle control group vs. treatment group: 2.50 ± 0.41 vs. 1.61 ± 0.42, P < 0.05; gelatinase, early control group vs. treatment group: 162 367 ± 5095 vs. 62 366 ± 2131, P < 0.01, middle control group vs. treatment group: 184 256 ± 5427 vs. 113 197 ± 4809, P < 0.01) while these parameters were similar between minocyclin-treated and control rats in late intervention group (all P > 0.05).</p><p><b>CONCLUSIONS</b>MMP-9 plays an important role in the pathogenesis of autoimmune myocarditis. Inhibition of MMP-9 in early and middle stage could significantly attenuate inflammatory responses and myocardial fibrosis in this experimental EAM model.</p>

Influence of cyclosporine A on atrial L-type calcium channel alpha1c subunit in a canine model of atrial fibrillation / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the impact of cyclosporine A (CsA) on atrial expression change of L-type calcium channel alpha1c subunit in a canine model of atrial fibrillation (AF).</p><p><b>METHODS</b>AF was induced by rapid atrial pacing (400 beats/min) for 8 weeks in adult male dogs and placebo (n = 6) or CsA (5 mg x kg(-1) x d(-1), n = 6) were orally administered to these animals. Sham operated animals served as normal controls (n = 6). The atrial electrophysiological parameters including P wave duration, atrial effective refractory period (AERP) were recorded and analyzed at baseline and 8 weeks later. Animals were scarified at 8 weeks post final electrophysiological examinations and atrial expressions of L-type calcium channel alpha1c subunit were determined by Western blot.</p><p><b>RESULTS</b>Compared to sham group, the P wave duration was significantly prolonged while AERP was significantly decreased in AF and CsA groups (all P < 0.05). AERP was significantly longer in CsA group than that in AF group (P < 0.05). L-type calcium channel alpha1c subunit expression was significantly downregulated in AF group compared to sham group (P < 0.05) and CsA significantly attenuated this downregulation (P < 0.01 vs. AF group).</p><p><b>CONCLUSION</b>CsA could attenuate the downregulation of the L-type calcium channel alpha1c subunit expression and improve the atrial electrophysiological remodeling in this canine model of AF.</p>