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13for expelling the heat; Rhizoma Typhonii, Angelica Dahurica, Spina Gleditsiae can neutralize the phlegm and remove the humidity, and they can also stop the pain. Rhizome of Chuanxiong and Safflower can invigorate blood circulation and removing blood stasis. Baicalein can also expel the heat and remove the humidity, which can also stanch and detoxicate. Rhubarb can help people defecate and expel the heat, which can also lower down the blood temperature and detoxicate. Tuckahoe, dried orange peel, and Tuber Pinellia can neutralize the phlegm and remove blood stasis, which can help the spleen improving its function. Sargassum, Thallus Eckloniae, and raw oyster can soften and remove the tuberculum, which is also good for the elimination of edema. All the medicines together can get twice the result with half effort. After successful recovery, recently it would appear the secondary pigmentation, but this symptom would gradually disappear between 3 months and half a year after all, and the skin would get back into its original color. After observation, this decoction has great effect on treating PSTA, which has not been found obvious side effect; this means it is much better than Zhongjiefeng Fensan Pill, which should be widely applied in clinical.
This disease is closely connected with the patient's diet, emotion, life pattern and so on, so docters should tell patients to keep healthy diet, life pattern, and positive emotion.
References
Bian Zhao (2001), Clinical Dermatology. [M]. Nanjing: Jiangsu Science and Technology Press, 2001:109.
Hongjian Huang (2012). From the "fire" on the Treatment of 80 Cases of Acne. [J].Henan: Henan Chinese Medicine, 35(1):34.
Jing Xue, and Pingmao Liang (2007), Acne treatment experience. [J].Sichuan: Sichuan Chinese Medicine, 25(7):7.
The State Administration of Traditional Chinese Medicine. The Criteria of Diagnosis and Therapeutic Effect of TCM. [S]. Nanjing: NanjingUniversity Press, 1994:158.
Total flavonoids from Hypericum attenuatum impact gene expression
of ion channels in arhythmic rats
Yangli WANG, Ji LI*
(Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, China 150040) *Corresponding Author (Ji LI) at: Heilongjiang University of Chinese Medicine, Heping Road 24, Xiangfang District, Harbin, Heilongjiang 150040, China Fax: +86 451 82936802; E-mail address: wangyanli.300@163.com(D.WANG) Keywords: Arrhythmic rats, total flavonoids,Hypericum attenuatum, CaL-a1C mRNA, Katp -K-6.1 mRNA
Summary. This study is an original one. We aim to elucidate the mechanism oftotal flavonoids from H. attenuatumand their antiarrhythmic effect in rats.H. attenuatum, is a well-knowntraditional Chinese medicine and is used primarily by medical scientists for the treatment of heart failure and arrhythmia. Thus, we anticipatethat people will recognizeour work, andthat this drug can be used inthe treatment of many patients.
Abstract
Studybasis: Our previousstudies have shown that the total flavonoids from Hypericum attenuatum have a potential function to treat arrhythmia. However, only a few studies have investigated the mechanism of total flavonoids from H. attenuatum to treat arrhythmia .
Objectives: In this study, we aim to elucidate the mechanism of antiarrhythmic action of total flavonoids from H. attenuatum.
Methods: Different groups of rat were fed total flavonoids or reference substance in advance. Arrhythmic rats were induced by carrying out the myocardial ischemia-reperfusion. Thereverse transcription-PCR (RT-PCR) method was used to detect the expression of CaL-a1C mRNA and KATP-Kir6.1 mRNA in the myocardial cell membrane of all groups of rat.
Results: The expressions of CaL-a1C mRNA and KATP -Kir6.1 mRNA on myocardial cell membrane in the experimental group significantly decreased compared with the other groups.
Conclusions: The results show that the total flavonoids from H. attenuatum have an important function in alleviating arrhythmia by impacting ion channels.
1.Introduction
Cardiac arrhythmias are common in the general population especially among patients with heart disease.In extreme cases, patients are susceptible to sudden death. Thus, continuous updating of knowledge in this field is needed1. In China, many traditional medicinesare used to treat arrhythmia includingH. attenuatum, whichis also known as metacentric grass. H. attenuatumis a perennial herb and is mainly distributed in specific regionsof China and other countries such as Korea, Japan, Mongolia, and Russian Far East.Our previous studies have shown that the flavonoids
2—4
from H. attenuatum have a potential function to treat arrhythmia .The purpose of this study is to observetheeffect of total flavonoids from H. attenuatumand the mechanisms involved intreating arrhythmic rats based on the expressions of CaL-a 1C mRNA and KATP-Kir6.1 mRNA. This study will serve as a foundation for the broader clinical application of this drug.
2.Experimental Methods
2.1 Equipment and Reagents
Gradient PCR (Eppendorf Corporation, Germany); the Medlab biological signal collecting and processing system (Nanjing Medease Science and Technology Co., Ltd, China); gel imaging system (Bio-Rad Company, USA); IBAS 2.5 full automatic image analysis system (Kontron Company, Germany); 6010 UV-vis spectrophotometer (American Agilent (Shanghai) Analytical Instrument Factory); DH-140 animal respirator (Taimeng Technology Co., Ltd., Chengdu, China); trace palm centrifuge (Germany SIGMA Company); refrigerator with a temperature of -80 °C (SANYO Company, Japan); desktop pH meter (Mettler Company, Switzerland); Primer Premier 5.0 (Primer Company,Canada); TaKaRa PrimeScript®RT-PCR Kit (TaKaRa Biotechnology (Dalian) Co.,Ltd.); TaKaRa DNA 100 Marker (TaKaRa Biotechnology (Dalian) Co., Ltd.);DNAGreen (10000x, Tiandz Genetic EngineeringCo., Ltd., China); agarose(Bioscience Company, Spain); primer synthesis (Shanghai Generay Biotech Co., Ltd, China). Allother experimental materials were from common commercial sources.
2.2 Animal Groupings
Adult Sprague-Dewey rats with a mean weight of 200 g (half of which were male and half were female) were obtained from the Drug Safety Evaluation Center of Heilongjiang University of Chinese Medicine, which is accredited by the Institutional Animal Care and Use Committee. All rats were provided with standard rat chowand water.
Thirty-six rats were randomly divided into three groups, namely, the conditional control, experimental, and untreated control groups. The experimental group was fed with H. attenuatumcontaining flavonoids of 100 mg/kg for 7 d by gavage, whereas the other two groups were provided with normal diet.
2.3 Model preparation
All animals were anesthetized by injecting of 25% urethane 5mL/kg while fixed face-up. The normal electrocardiogram (ECG) was recorded until a stable state that lasted for 15 min was observed. The median cervical skin was then cut and opened, and the trachea was exposed via blunt dissection. The center of the trachea was then cut with a T-type hole at the center, and a plastic pipe with a diameter of 2 mm was inserted. This pipe was used for artificial respiration by an animal
respirator with a ventilation of 8 to 9 mL/100 g and a respiratoryrate of 50 to 60 times/min. The ratio of inhalation to that of exhalation was 2:1. The respiratorwas set to take a continuous positive pressure breathing of 4 to 5 kPa.
The skin of the left sternal border was longitudinally cut with a length of about 2 cm and opened. The subcutaneous tissues and muscles were separated layer by layer, and the chest was opened in the intercostal area where the beating of the heart was most obvious. The costa by eye speculum was dilated. The eye speculum after unriping the pericardium was pressed down to make the heart pop-up from the chest. The left main coronary artery located between the left atrium and the right pulmonaryconus5 was inserted with a 5/0 atraumatic suture needle through the myocardial surface, which was then twisted beneath theleft anterior below the coronary artery and removed from the other side. A slipknot was made to ligate the left anterior below the coronary artery. The slipknot was then opened after 30 min. A dark color of the myocardial surface under the ligation point indicated ischemia, whereas a red color indicated ischemia-reperfusion6.
After ischemia-reperfusion, the rib,muscle and skin were sutured layer by layer. The breathing of the rats was ensured via the respirator butwith decreased ventilation for 60 min. The conditional control group had the same operation procedure except ligation was not performed.
Throughout the entire surgical procedure, the author has to abandon rats in which ligation or reperfusion were not successful, bled excessively, or had a respiratory and cardiac arrest after more than 30 s. A total of three rats were abandoned from the experimental group and onefrom the untreated control group.
2.4 Tissue Preparation
After preparing the model, rat heart was quickly removed and flushed with saline water. Subsequently, the heart was dried multiple times by using filter paper, transferred in a freezing tube, labeled,and immediately placedin liquid nitrogen. Full-out was performed when needed.
2.5 Detection of mRNA by RT-PCR
2.5.1 Total RNA extraction
1) About 100 mg of the sample was transferred in a precooled mortar, groundafterthe addition of liquid nitrogen, and placed in a 1.5 mL eppendorf (EP) tube; 2) About 1 mL oftrizol was added in the EP tube, blended upside down for ten times, and was allowed to stand for 5 min at room temperature; 3) About 200 ^L of CCl4 was added and vigorously vibrated for 15 sec and allowed to stand for 2 to 3 min at room temperature; 4) The sample was centrifuged at 12000 g/min with a temperature of4 °C for 15 min, and about 500 ^L of the extracted supernatantwas transferred to another 1.5 mL EP tube; 5) The supernatant was added with 500 ^L of isopropanol, blended upside down, and allowed to stand at room temperature for 10 min; 6) The sample was again centrifuged at 13000 g/min with a temperature 4 °C for 10 min. Thesupernatant was removed,and the precipitate was dried using absorbent paper; 7) About 1 ml of cold 75%ethanol was added to wash the precipitate; 8) The sample was centrifuged at 10000 g/min with a temperature 4 °C for 10 min. The supernatant was removed, and the precipitate was dried using absorbent paper; 9) The precipitate was allowed to dry in air for 10 min; 10) About 30 ^L of diethypyrocarbonate (DEPC) water was used to dissolve the precipitate; and 11) the total RNA was measured using a spectrophotometer.
2.5.2 Experimental procedures of reverse transcriptase (synthetic cDNA) 3. Preparation of the reverse transcriptase reaction solution: 5xExScriptTM Buffer2.0 |L
dNTP Mixture (10mMeach)0.5|L OligodTPrimer(50|M) 0.5 |L PrimeScriptTMReverseTranscriptase (200U/|L)0.25|L RNase Inhibitor (40 U/|L) 0.25|L
Total RNA 500 ng RNase Free ddH2Oup to 10 |L.
2) Total: 10 ^L/sample.
3) Reverse transcription reaction in the following conditions:
42 °C30 min 99 °C5 min 5 °C5 min
Operated in the above conditions for one cycle.
4) Primer design
P-actin (The amplified fragment is 546 bp): P-actin-EP: 5' -AAATGCTGGTGACATCAAA-3' P-actin-RP: 5' -AAGAAAGGGTGTAAAACGCA-3' CaL-a1C (The amplified fragmentis 793 bp): CaL-a1C-EP: 5'-AGAGCAAAAGCTCAAATTCACTG-3' CaL-a1C-RP: 5'-ACTTTTAAAAATGCTTCCACGGT-3' KATP-Kir6.1 (The amplified fragment is 460 bp): KATP-Kir6.1-EP: 5'-TGATCATCTGCCATGTGATTGAT-3' KATP- Kir6.1- RP: 5'-TTTCCTTCTGGAGTCATGAATTG-3' 2.5.3 PCR reaction
1) Preparation of the PCR reaction solution
5xPCR Buffer10 p,L sterile distilled water28.75 ^L TaKaRa ExTaq HS0.25 p,L Primer 10.5 p,L Primer 20.5 p,L
2) Total: 40 ^L/sample
3) PCR reaction conditions 94 °C 2 min
94 °C 30 sec 54 °C 30 sec 72 °C 2 min 72 °C 7 min Operated in the above conditions for 30 cycles.
2.5.4 Agarose gel electrophoresis
The 1.0% agarose gel with 0.5 g of agarose and 50 mL of 1*TAE were prepared.The gel was dissolved in a microwave, cooled to 55 °C, 5 ^L of DNA Green was added. The comb was removed 30 min after. The gum was placed in the electrophoresis buffer.
The sample was then mixed. About 10 ^L of the sample plus 2 ^L of 10* loading buffer was placed in a glue hole. About 5 ^L of DL2000 marker was used as the control sample. The samples were electrophoresed at 110 V for 30 min.
The analysis, scanning, and photography were achieved using a cohesion imaging system. The date of average density and the band areawere obtained. Gene expression was calculated using the formula as follows: Gene expression=(measured intensity of the target gene*area of the target gene)/( intensity of P-actin*area of P-actin).
2.5.5 Statistical analysis
Data analysis was performed using Microsoft Excel 2000 and Clampfit 8.2 (Axon Instruments, Foster City, CA, USA). Mean ± standard deviation was used throughout the text. A
two-tailed unpaired t-test with equal variances was used in the estimation of statistical significance of the differences, which were considered significant if P<0.05. 3.Experimental results
3.1 The expression of CaL-a1C mRNA in the conditional control group is normal(0.210±0.008).Nevertheless, the experimental group animals and the conditional control group have a significantly lower expression of CaL-a1CmRNA (0.367±0.030)compared with the untreated control group (0.766±0.036)(P<0.01).(Fig.1andFig.2)
Fig.1. Effects of total flavonoids from H. attenuatumon the expression of CaL-aiC mRNA in the cell membrane of myocardial ischemia-reperfusion arrhythmic rats
The expression of CaL-txlc mRNA
Note: **P<0.01 compared with the Untreated control group. Fig.2. Effects of total flavonoids from H. attenuatum on the expression of CaL-aic mRNAinthe cell membrane of myocardial ischemia-reperfusion arrhythmic rats
Note: M.DNA Marker;
1. Conditional control group;
2.Untreated control group;
3.Experimental group
3.2 The expression of KATP-Kir6.1 mRNA in the conditional control group is normal(0.435±0.046).Nevertheless, the total flavonoids from H. attenuatum can reduce theexpression of KATP-Kir6.1 mRNA inthe cell membrane of myocardial ischemia-reperfusion arrhythmicrats (0.591±0.018) compared with the untreatedcontrol group (1.045±0.037). The difference is statistically significant (P<0.01). (Fig. 3 and Fig.4.)
4. Discussion
L-type calcium (Ca2+) channel is composedofa1c, a2S, and P2.a1c has a holein which Ca2+ ions enter the cell ,and is the major subunit that determines the voltage of dependent Ca channel and drug susceptibility. In this experiment,the high level expression of the a1c subunit mRNA indicates that the L-type calcium channel is highly open and results in more Ca2+ ions to enterthe cell. Studies have shown that the increased expression of L-type Ca channel or extension of opening hourscan prolong action potential duration (APD); thus, the level of the plateau potential increases, the early stage of after-depolarization is induced, and the after-depolarizationand other triggered arrhythmia are delayed8. The ATP-sensitive potassium channel (KATP) isweak in the rectifier potassium (K) channel, which is regulatedby ATP concentration in the cells. It also is a kind
ofmembrane-spanning ion channel that is widelydistributed in a variety of cells and organelles,coupling cell excitabilityand energy metabolism, and hasbiological and pathological significance in organizations.
Fig.3 Effects of total flavonoids from H. attenuatum on the expression of KATP-Kir6.i mRNA in thecell membrane of myocardial ischemia-reperfusion arrhythmic rats
1.2 r
The expression of KATP-Kirfi.l mRNA
Note: **P<0.01 compared with the Untreated control group. Fig.4 Effects of total flavonoids from H. attenuatum on the expression of KATP-Kir6.i mRNAinthe cell membrane of myocardial ischemia-reperfusion arrhythmic rats
M 1 2 3 M I 2 3
KATP is an eight polymer molecule and is composed of two channel subunits.Onechannel subunitis known as Kir6.x, which is aninward rectifier subunit that includes Kir6.1 and Kir6.2. The other subunit is a regulatory subunit known as sulfonylurea receptor,SURx.Kir6.x forms the center hole of the KATP channel.Kir6.1 exists in the myocardial cell membraneand in the myocardial mitochondrial membrane.Morrisseyetal. found thatKir6.1 expresses on the ventricular muscle cell membrane of mice and rats viaimmune localization9.Researchers have performed ligation on the left coronary artery ofrat hearts to mimic ischemia-reperfusion injury. Theyfound that the expression of Kir6.1 subunitmRNA and protein in the ischemic and non-ischemic areassignificantly increased than the blank controlgroup. However, Kir6.2 did not significantly change.10 Another in vivo study has also shown that the expression of Kir6.1 subunit mRNA in the hypoxia groupis higher than in the normal group, but the expression of Kir6.2 decreased11.
The results of the present study show that the total flavonoidsfrom H. attenuatumcan reduce the cell membrane expression of CaL-a1CmRNA inmyocardial ischemia-reperfusion arrhythmicrats but increase the expression of KATP-Kir6.1mRNA.Thus, H. attenuatumcandirectly
or indirectly function in the L-type Ca channel and in the KATP in myocardial cells.H. attenuatuminhibitsthe inflow of Ca2+ions and regulatesthe Ca2+ion concentration in myocardial cells by inhibiting the height of the opening of L-type Ca channels. Moreover, H. attenuatumrestrains the occurrence of tachyarrhythmia by facilitatingthe opening of KATP. Further studyisrequired to clarify the mechanism of flavonoidsofH. attenuatumagainstmyocardial ischemia-reperfusion arrhythmia.
5.Acknowledgements
This work was supported by National Natural Science Foundation of China(81173185); Key Project of Natural Science Foundation of Heilongjiang Province(ZD201111); Heilongjiang Province Graduate Students ResearchKey Project (YJSCX2012-334HLJ); and Outstanding Talents Support Program of Heilongjiang University of Chinese Medicine(2012).
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Studies on Improving Diosgenin Content of Dioscorea zingibenisis with Solid
Fermentation of Endophytic Fungi C39
Ying Xu, Siqi Gu, Dan Yu, Xiaowei Du
Pharmacognosy,HeilongjiangUniversity of Chinese Medicine, Harbin,China
Abstracts
Objectives: The aim of the study was to use solid fermentation of C39 isolated from dioscorea nipponica improving the content of diosgenin in D.zingibenisis C.H. Wright to find the
