1.RNA SNP Detection Method With Improved Specificity Based on Dual-competitive-padlock-probe
Qin-Qin ZHANG ; Jin-Ze LI ; Wei ZHANG ; Chuan-Yu LI ; Zhi-Qi ZHANG ; Jia YAO ; Hong DU ; Lian-Qun ZHOU ; Zhen GUO
Progress in Biochemistry and Biophysics 2024;51(11):3021-3033
ObjectiveThe detection of RNA single nucleotide polymorphism (SNP) is of great importance due to their association with protein expression related to various diseases and drug responses. At present, splintR ligase-assisted methods are important approaches for RNA direct detection, but its specificity will be limited when the fidelity of ligases is not ideal. The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection. MethodsIn this study, a dual-competitive-padlock-probe (DCPLP) assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation. To verify the method, we employed dual competitive padlock probe-mediated rolling circle amplification (DCPLP-RCA) to genotype the CYP2C9 gene. ResultsThe specificity was well improved through the competition and strand displacement of dual padlock probe, with an 83.26% reduction in nonspecific signal. By detecting synthetic RNA samples, the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L. Furthermore, clinical samples were applied to the method to evaluate its performance, and the genotyping results were consistent with those obtained using the qPCR method. ConclusionThis study has successfully established a highly specific direct RNA SNP detection method, and provided a novel avenue for accurate identification of various types of RNAs.
2.Effect and Mechanism of Xiangsha Liujunzi Tang on Lipid Deposition in Liver of ApoE-/- AS Mice by Affecting Long Noncoding RNA-HC/miR-130b and to Regulate Cholesterol Metabolism
Si CHEN ; Nan SONG ; Ying WANG ; Hui-min CAO ; Xiao YANG ; Fang YANG ; Qun WANG ; Lian-qun JIA
Chinese Journal of Experimental Traditional Medical Formulae 2021;27(3):15-21
Objective:To investigate the mechanism of Xiangsha Liujunzi Tang in improving liver lipid deposition in ApoE-/- atherosclerotic (AS) mice by affecting long noncoding RNA-HC (Lnc-HC)/microRNA-130b (miR-130b) in the regulation of cholesterol metabolism. Method:Totolly 10 C57BL/6J mice were selected as normal controls, and 30 healthy ApoE-/- mice fed with high fat diet for 12 weeks were then randomly divided into the model group, Xiangsha Liujunzi Tang group(19.12 g·kg-1·d-1) and simvastatin group(2.275 mg·kg-1·d-1), with gavage administration for 4 weeks. The serum lipid level of mice was detected by automatic biochemistry analyzer, and the histopathological changes of liver cells were observed by hematoxylin-eosin (HE) staining. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect expression of long noncoding RNA-HC, and miR-130b. Real-time PCR and Western blot assay were used to detect gene and protein expression of peroxisome proliferator-activated receptor gamma (PPAR
3.Study on Inhibitory Effect of Huayu Qutan Recipe on Myocardial Fibrosis in Atherosclerotic Rabbits Based on Mitochondrial Fusion-lysis
Qu ZHENG ; Lian-qun JIA ; Nan SONG ; Ning CHEN ; Si CHEN ; Guan-lin YANG
Chinese Journal of Experimental Traditional Medical Formulae 2020;26(3):71-77
Objective::To analysis the effect of Huayu Qutan recipe on myocardial fibrosis in atherosclerotic rabbits based on mitochondrial fusion-lysis. Method::The 36 SPF healthy male rabbits were selected, and 6 rabbits were selected randomly as the normal group, and given normal pellet feed, another 30 rats were fed with high fat diet to establish atherosclerosis model.After successful replication of animal models, they were randomly divided into model group, Huayu Qutan recipe low-dose, medium-dose and high-dose group (4.0, 8.0, 16.0 g·kg-1) and simvastatin group (1.4 mg·kg-1), 6 rats each.Each group was given corresponding drugs according to the dosage, continuous administration for 4 weeks.The serum lipid levels in rabbits of each group were detectived by automatic biochemical analyzer, the degree of myocardial fibrosis was measured by Masson staining, and expression levels of mitochondrial fusion protein 1 (Mitofusin 1), mitochondrial fusion protein 2 (Mitofusin 2), optic atrophy protein 1 (Opa1), promoter protein 1 (Drp1), mitogen 1 (Fis1) in myocardial tissue were detected by immunohistochemistry. Result::Compared with normal group, the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) in model group were significantly increased, levels of density lipoprotein cholesterol (HDL-C) were significantly decreased, the expression levels of Mitofusin 1, Mitofusin 2 and Opa1 in myocardial tissue were significantly decreased, the expression levels of Drp1 and Fis1 were significantly increased(
4.Potential toxic effects of TDCIPP on the thyroid in female SD rats.
Jing Ran SUN ; Mi Mi FAN ; Zhen ZHANG ; Jin WU ; Dian Peng HAN ; Jia Lei BAI ; Lian Qun DU ; Yan Jun FANG
Chinese Journal of Applied Physiology 2020;36(3):250-254
To investigate the potential toxic effects and mechanisms of Tris(1; 3-dichloro-2-propyl) phosphate (TDCIPP) on thyroid in female SD rats. Thirty-two 3-weeks-old female SD rats were randomly divided into normal group(treated with corn oil ), and low/moderate/high-dose group treated with TDCIPP (dissolved in corn oil )(n=8). All rats were treated with corn oil or TDCIPP (50, 100, 250 mg/(kg·d)) once a day during a 21-day period. All rats were sacrificed after the last administration. Serum thyroid stimulating hormone (TSH), 3,3',5-triiodothyronine (T3), 3,3',5,5'-tetraiodothyronine (T4), free 3,3',5,5'-tetraiodothyronine (FT4) were detected with ELISA kit. Morphology of thyroid was observed with hematoxylin and eosin (HE) staining. Expressions of genes and proteins correlate with thyroid were measured respectively by real-time fluorescence quantitative PCR and Western blot. Compared with control group, morphology of thyroid showed follicles irregular arrangement, hypocolloid, and follicular hyperplasia in TDCIPP treatment groups. The levels of serum TSH in low-dose TDCIPP group and T3 in high-dose TDCIPP group were significantly higher than those in control group(P<0.05). Thyroid stimulating hormone receptor (TSHR) mRNA expression was decreased distinctly in low-dose TDCIPP group, while the expression of thyroperoxidase (TPO) mRNA was increased notably in moderate and high-dose TDCIPP groups(P<0.05,P<0.01). Compared with control group, the level of TRβ protein was decreased significantly in moderate and high-dose TDCIPP groups, while the expressions of udp-glucuronosyl-transferases (UGTs) and cytochrome-p450-3A1 (CYP3A1) proteins were upregulated notably in TDCIPP treatment groups(P<0.05). Treated with 50 mg/(kg·d) TDCIPP can cause thyroid hyperplasia, change the levels of thyroid hormones, and disturb thyroid function, therefore, it has toxic effects on the thyroid.
5. Interpretation of Prescription and Syndrome of Yangjing Zhongyu Tang and Its Modern Research Progress
Jun-ping ZHU ; Jia-ming WEI ; Rui-lian LIU ; Hui-hui LIANG ; Yan MAO ; Wen WEN ; Ge YU ; Yi-qun ZHOU ; Fu-yuan HE ; Wen-long LIU ; Xi-li ZHANG
Chinese Journal of Experimental Traditional Medical Formulae 2019;25(23):209-214
Yangjing Zhongyu Tang, from FU Qing-zhu Nvke, is a famous classical formula of clinical value for treating blood deficiency syndrome of female infertility in the Qing dynasty. The prescription seems plain, but it is rigorous and effective with high research value. In this paper, the ancient books and modern documents of Yangjing Zhongyu Tang were analyzed from the aspects of traceability of Chinese materia medica in the formula, pharmacological research, clinical research, etc. It could be concluded that the pharmacological studies of this formula were mostly to investigate the mechanism and efficacy of its treatment for infertility, but there was a lack of comprehensive interpretation of the structure, function and principle of pharmacodynamic substance in this formula. In this paper, combined with the contents of supramolecular imprinting template and network pharmacology, the new direction of pharmacological research of Chinese medicine compound was put forward. Quality control of Yangjing Zhongyu Tang mostly based on small molecule compounds, so it was suggested to break the inherent thinking, and increase the detection of macromolecule compounds and supramolecular of Chinese materia medica. The druggability of this formula involved in the preliminary study of decoctions, plasters and granules. Considering the quality stability of Chinese materia medica in the formula and the scientificity of druggability of this formula, the compatibility principle of traditional Chinese medicine (TCM) and modern supramolecular chemistry theory could be combined to study the change law of druggability of TCM before and after compatibility, so as to provide new reference materials for the follow-up clinical application and development of Yangjing Zhongyu Tang.
6. Analysis on “Point-line-surface” Quality Standard of Yangjing Zhongyu Tang
Jun-ping ZHU ; Jia-ming WEI ; Rui-lian LIU ; Hui-hui LIANG ; Ge YU ; Wen WEN ; Yan MAO ; Yi-qun ZHOU ; Wen-long LIU ; Fu-yuan HE ; Xi-li ZHANG ; Long LI
Chinese Journal of Experimental Traditional Medical Formulae 2019;25(21):12-18
Objective: Based on the analysis of the total components of fingerprint and the determination methods of the existing components in the 2015 edition of Chinese Pharmacopoeia, the "point-line-surface" quality standard of Yangjing Zhongyu Tang was established by the "point" of each single component (morroniside, loganin, paeoniflorin, ferulic acid and verbascoside) to the "line" of multicomponent and the "face" of fingerprint of the whole component. Method: XB-C18 column (4.6 mm×250 mm, 5 μm) was used for gradient elution of 0.1% phosphoric acid aqueous solution-acetonitrile. The column temperature was 30℃, the injection volume was 10 μL, the flow rate was 1.0 mL·min-1, and the detection wavelengths were 240, 316, 230, 334 nm. The contents of these five components in Yangjing Zhongyu Tang were determined by three correction methods, external standard method and regression equation method. At the same time, the fingerprint of Yangjing Zhongyu Tang were analyzed by total component analysis and similarity evaluation. Result: With ferulic acid as reference, the relative correction factor (f) of morroniside, loganin, paeoniflorin and verbascoside were 0.392 1, 0.421 4, 0.261 7, 0.268 6 by multi-point correction method, and their f (slope correction method) were 0.385 4, 0.419 4, 0.255 9, 0.274 0, respectively. Twenty characteristic peaks of fingerprint were analyzed and the similarity was ≥ 0.999.There was no significant difference in the contents of these five components from Yangjing Zhongyu Tang determined by the quantitative assay of multi-components by single-marker (QAMS) correction method, the external standard method and the regression equation method. Conclusion: The comprehensive quality standard established by the total component analysis of fingerprint combined with various determination methods of existing components in the 2015 edition of Chinese Pharmacopoeia has been validated in famous classical formula of Yangjing Zhongyu Tang, which can provide ideas and methods for the quality control with quantitative determination and fingerprint of other famous classical formulas.
7. Effect of Xiangsha Liu Junzitang on dyHDL in Hyperlipidemia Rats with Spleen Deficiency
Si CHEN ; Lian-qun JIA ; Nan SONG ; Jian WANG ; Ning CHEN ; Yuan CAO ; Yun-fei GAO ; Xiao-ming LYU
Chinese Journal of Experimental Traditional Medical Formulae 2019;25(7):22-28
Objective: To observe the changes of dysfunctional high density lipoprotein cholesterol (dyHDL) and the intervention effect of Xiangsha Liu Junzitang in rats with spleen deficiency and hyperlipidemia, and reveal the effect and mechanism of Xiangsha Liu Junzitang on dyHDL in rats with spleen-deficiency hyperlipidemia. Method: Seventy-five SPF SD rats were randomly divided into normal group, high fat group, spleen deficiency and high fat group, Xiangsha Liu Junzitang low and high dose groups (5.67, 11.34 g·kg-1). In the spleen deficiency and high fat group, as well as Xiangsha Liu Junzitang low and high dose groups, composite method of improper diet and exhaustive swimming was used for 15 days for modeling. After modeling for 15 days, normal group was fed with basic diet, while the high-fat group, spleen-deficiency and high-fat group, the Xiangsha Liu Junzitang low and high dose groups were fed with high-fat diet. After 12 weeks, the Xiangsha Liu Junzitang low dose and high dose groups received corresponding dosage of drugs, while normal group, high fat group and spleen deficiency high fat group received corresponding volume of normal saline. After 4 weeks, the contents of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol cholesterol (LDL-C), and high density lipoprotein cholesterol(HDL-C)were detected by automatic biochemistry analyzer, while D-xylose excretion rate was measured by phloroglucinol method. The morphological changes of liver cells were observed by hematoxylin eosin (HE) staining. The level of PON1, apoA1 and SAA in plasma were detected by enzyme-linked immunosorbent assay (ELISA). Paraoxonase 1(PON1), apolipoprotein A1 (apoA1) and serum amyloid protein A (SAA) gene expression in rats liver were detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). Result: As compared with normal group, the serum TC, TG, and LDL-C levels were significantly increased in the high-fat group and spleen-deficiency high-fat group (P<0.05), and HDL-C levels were significantly lower (P<0.05); the levels of PON1 and apoA1 in plasma were decreased (P<0.05), while the content of SAA was increased (P<0.05); the expression of SAA gene in liver tissues was increased (P<0.01), and the expression levels of PON1 and apoA1 genes in liver tissues were significantly decreased (P<0.01). In high-fat group and the spleen-deficiency and high-fat group, the hepatocytes were rounded and scattered, and scattered fat vacuoles were observed. In addition, the urinary D-xylose excretion rate was significantly decreased in the spleen-deficiency and high-fat group (P<0.05). After the intervention with Xiangsha Liu Junzitang, the serum TC, LDL-C levels were significantly decreased (P<0.05); HDL-C levels were significantly increased (P<0.05); plasma PON1, apoA1 levels were increased (P<0.05); the content of SAA was decreased (P<0.05); the expression of SAA gene in liver tissues was decreased (P<0.05); the expression of apoA1 gene was increased significantly (P<0.05). Liver cells swelling was significantly alleviated and fat foaming was reduced. As compared with high-fat group, the plasma PON1 and SAA levels in the spleen-deficiency high-fat group were significantly lower (P<0.05); hepatocyte swelling was obvious and foaming was aggravated. Conclusion: The lipid disorder in hyperlipidemia rats was aggravated by the spleen deficiency, but was corrected after intervention with Xiangsha Liu Junzitang. and its mechanism may be related to the regulation of the expression of dyHDL-related genes and protein.
8. Phenolic Components from Roots of Psidium guajava
Sheng-jia CHEN ; Ying-zheng HUANG ; Jian LU ; Jian-qun LIU ; Hui-lian HUANG ; Ji-cheng SHU
Chinese Journal of Experimental Traditional Medical Formulae 2019;25(2):169-174
Objective:The different parts of Psidium guajava(Myrrhinaceae) have different bioactivities. There are intensive studies for chemical constituents of its leaves and fruits at present. However,there are a few studies for the roots. Therefore, we systematically investigated the chemical constituents of phenolic acids in roots of P.guajava. Method:The chemical constituents were isolated and purified by various column chromatography methods and semi-preparative HPLC. The isolated compounds were identified by physicochemical properties and spectral analysis. Result:Sixteen phenolic components were isolated from the ethanol extract and identified as 3,3',4'-tri-O-methylellagic acid(1),3,3',4-tri-O-methylellagic acid-4'-O-β-D-glucopyranoside(2),3-O-methylellagic acid-4'-O-α-L-rhamnopyranoside(3),3'-O-methyl-3,4-O,O-metheneellagic acid-4'-O-β-D-glucopyranoside(4),gallic acid(5),methyl gallate(6),ethyl gallate(7), 3,4,5-trimethoxypheny-1-β-D-glucopyranoside(8),3,5-dimethoxy-4-hydroxy-benzoic acid-7-O-β-D-glucoside(9),1-hydroxy-3,4,5-trimethoxyphenyl-1-O-[6'-O-(4″-carboxy-1″,3″,5″-trihydroxy)phenyl]-β-D-glucopyranoside(10),vanillic acid(11),protocatechuic acid(12),secoisolariciresinol 9-O-β-D-glucopyranoside(13),phloretin 4'-O-β-D-glucopyranoside(14),cinchonain Ib(15) and epicatechin(16). Conclusion:Compounds 3,4,6,8-10,13-15 were isolated from this plant for the first time.
9.Relationship Between the Changes of Left Atrium, Pulmonary Vein Ostia and AF Recurrence After Radiofrequency Catheter Ablation by Enhanced Cardiac MRI Evaluation
Xue-Lian LI ; Wen-Su CHEN ; Cheng-Zong LI ; Fei LI ; Chao-Qun ZHANG ; Shu-Guang HAN ; Jia-Li WANG ; Zhi-Rong WANG ; Zhuo-Qi ZHANG
Chinese Circulation Journal 2018;33(4):390-394
Objectives: To evaluate the changes of left atrial volume (LAV) and the maximum ostial cross-sectional area (CAS) of pulmonary vein (PV) in atrial fibrillation (AF) patients after circumferential pulmonary vein isolation radiofrequency catheter ablation (CPVA-RFCA) and to explore their relationship to AF recurrence by enhanced cardiac MRI evaluation. Methods: Our research included in 2 groups: Control group, n=20 healthy subjects and AF group, n=78 patients whom were classified into 2 subgroups as Paroxysmal AF subgroup, n=46 and Persistent AF subgroup, n=32; 66 patients received CPVA-RFCA and based on 6 months post-operative recurrence, they were divided into another set of 2 groups: AF recurrent subgroup, n=17 and Non-AF recurrent subgroup, n=49. Pre- and 6 months post-operative maximum ostial CSA of PV were measured by enhanced cardiac MRI, LAV were obtained by 3D reconstruction and the differences were compared between AF group and Control group, Paroxysmal AF subgroup and Persistent AF subgroup, AF recurrent subgroup and Non-AF recurrent subgroup; their relationships to AF recurrence were studied.Results: Compared with Control group, AF group had increased LAV and elevated ostial CSA of superior PV (SPV), both P<0.05. Compared with Paroxysmal AF subgroup, Persistent AF subgroup had increased LAV and elevated ostial CSA of SPV, both P<0.05. Compared with pre-operative condition, at 6 months after the operation, Non-AF recurrent subgroup showed reduced ostial CSAs in left SPV (LSPV), right SPV (RSPV), right inferior PV (RIPV) and decreased LAV, all P<0.05;while AF recurrent subgroup showed expanded RSPV and increased LAV,allP<0.05.Post-operative reductions of LAV and ostial CSA of SPV had close correlation; multivariate Logistic regression analysis indicated that LAV (HR=1.05, P<0.01)and ostial CSA of RSPV(HR=1.09,P=0.05)were related to AF recurrence after RFCA. Conclusions: CAPV-RFCA could reverse left atrial and PV remodeling in AF patients, LAV and ostial CSA of RSPV were related to post-operative AF recurrence.
10.Clinical features analysis of Qingkailing injection for adolescent patients based on real world HIS.
Li-Zhen CHEN ; Yan-Ming XIE ; Lian-Xin WANG ; Yin ZHANG ; Qun WANG ; Ping-Ping JIA ; Bo FENG
China Journal of Chinese Materia Medica 2017;42(15):2864-2870
To explore the clinical application features of Qingkailing injection for adolescent patients. This study was based on the information from the hospitalized patients in 1998-2011 in HIS (hospital information system) of 16 tertiary hospitals in China. 615 adolescent patients with Qingkailing injection were used to investigate the clinical characteristics of Qingkailing injection. Apriori algorithm was adopted to establish the model, and Clementine 12.0 was used for correlation analysis. The results showed that male patients (355 cases) were more than female patients (243 cases); the age of the most patients was under 14 years old, with an average age of 9.40 years old; the hospital stay was most of 4-7 d; the patients were mostly concentrated in pediatrics, mainly including upper respiratory tract infection; outpatient admission to the majority (451 cases, accounting for 75.54%). The traditional Chinese medicine (TCM) syndrome mainly included phlegm-dampness internal resistance (38.46%), Qi and Yin Deficiency (11.54%), Liver qi stagnation (15.38%), and Yang deficiency syndrome (11.54%); the highest admission rate was during Slight Cold (13.01%). In combined application, the most common western medicine pair was Cephalosporin injection+vitamin C (with supporting rate of 24.6%) and the most common TCM pair was Shuanghuanglian+Ganmao Qingre granules (with supporting rate of 4.065%). The most common 3 western medicines in combined use were Potassium chloride injection+Cephalosporin injection+vitamin C (supporting rate of 15.93%); while the most common three Chinese medicines were Shuanghuanglian+Yunnan Baiyao+Ganmao Qingre granules (supporting rate of 1.138%). Qingkailing injection was also widely used in adolescent patients; the diagnosis and use were basically in line with the instructions; men were slightly more than women; age of onset, solar terms and TCM syndromes were highly consistent. Qingkailing injection was mainly combined with western medicines in combined drug use, and Chinese medicines were also available. Combined drug use was more reasonable. Based on the results of the real world HIS, Qingkailing injection could provide the idea and reference for regulating the medication in adolescent patients.

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