RNA-Seq Profiling to Investigate the Mechanism of Qishen Granules on Regulating Mitochondrial Energy Metabolism of Heart Failure in Rats.

Background. Qishen granules (QSG) are a frequently prescribed formula with cardioprotective properties prescribed to HF for many years. RNA-seq profiling revealed that regulation on cardiac mitochondrial energy metabolism is the main therapeutic effect. However, the underlying mechanism is still unknown. In this study, we explored the effects of QSG on regulating mitochondrial energy metabolism and oxidative stress through the PGC-1α/NRF1/TFAM signaling pathway. RNA-seq technology revealed that QSG significantly changed the differential gene expression of mitochondrial dysfunction in myocardial ischemic tissue. The mechanism was verified through the left anterior descending artery- (LAD-) induced HF rat model and oxygen glucose deprivation/recovery- (OGD/R-) established H9C2 induction model both in vivo and in vitro. Echocardiography and HE staining showed that QSG could effectively improve the cardiac function of rats with myocardial infarction in functionality and structure. Furthermore, transcriptomics revealed QSG could significantly regulate mitochondrial dysfunction-related proteins at the transcriptome level. The results of electron microscopy and immunofluorescence proved that the mitochondrial morphology, mitochondrial membrane structural integrity, and myocardial oxidative stress damage can be effectively improved after QSG treatment. Mechanism studies showed that QSG increased the expression level of mitochondrial biogenesis factor PGC-1α/NRF1/TFAM protein and regulated the balance of mitochondrial fusion/fission protein expression. QSG could regulate mitochondrial dysfunction in ischemia heart tissue to protect cardiac function and structure in HF rats. The likely mechanism is the adjustment of PGC-1α/NRF1/TFAM pathway to alleviate oxidative stress in myocardial cells. Therefore, PGC-1α may be a potential therapeutic target for improving mitochondrial dysfunction in HF.

Effect of DanQi Pill on PPARα, lipid disorders and arachidonic acid pathway in rat model of coronary heart disease.

BACKGROUND: Danqi pill (DQP) is one of the most widely prescribed formulas and has been shown to have remarkable protective effect on coronary heart disease (CHD). However, its regulatory effects on lipid metabolism disorders haven't been comprehensively studied so far. We aimed to explore the effects of DQP on Peroxisome Proliferator activated receptors α (PPARα), lipid uptake-transportation-metabolism pathway and arachidonic acid (AA)-mediated inflammation pathway in rats with CHD. METHODS: 80 Sprague-Dawley (SD) Rats were randomly divided into sham group, model group, positive control group and DQP group. Rat model of CHD was induced by ligation of left ventricle anterior descending artery and fed with high fat diet in all but the sham group. Rats in sham group only underwent thoracotomy. After surgery, rats in the positive control and DQP group received daily treatments of pravastatin and DQP respectively. At 28 days after surgery, rats were sacrificed and plasma lipids were evaluated by plasma biochemical detection. Western blot and PCR were applied to evaluate the expressions of PPARα, proteins involved in lipid metabolism and AA pathways. RESULTS: Twenty eight days after surgery, dyslipidemia developed in CHD model rats, as illustrated by elevated plasma lipid levels. Expressions of apolipoprotein A-I (ApoA-I), cluster of differentiation 36 (CD36) and fatty acid binding protein (FABP) in the heart tissues of model group were down-regulated compared with those in sham group. Expressions of carnitine palmitoyl transferase I (CPT-1A) and lipoproteinlipase (LPL) were also reduced significantly. In addition, levels of phospholipase A2 (PLA2) and cyclooxygenase 2 (COX-2) were up-regulated. Expressions of Nuclear factor-κB (NF- κB) and signal transducer and activator of transcription 3 (STAT3) also increased. Furthermore, Expression of PPARα decreased in the model group. DQP significantly up-regulated expressions of ApoA-I and FABP, as well as the expressions of CPT-1A and CD36. In addition, DQP down-regulated expressions of PLA2, COX-2 and NF-κB in inflammation pathway. Levels of STAT3 and LPL were not affected by DQP treatment. In particular, DQP up-regulated PPARα level significantly. CONCLUSIONS: DQP could effectively regulate lipid uptake-transportation-metabolism process in CHD model rats, and the effect is achieved mainly by activating ApoA-I-CD36-CPT-1A molecules. Interestingly, DQP can up-regulate expression of PPARα significantly. The anti-inflammatory effect of DQP is partly exerted by inhibiting expressions of PLA2-COX2 -NF-κB pathway.

[Experimental study on relationship between pungent-hot herb property express and calmodulin].

OBJECTIVE: To explain the essence of pungent-hot herb property express according to in vivo and in vitro studies on its effect on calmodulin on the base of the observation of the adjustment in hypothalamic-pituitary-gonad axis functions of Aconiti Lateralis Radix Praeparata, Curculiginis Rhizoma, Cinnamomi Cortex and bitter-cold herb Phellodendri Chinensis Cortex in rats under the state of yang deficiency. METHOD: The yang-deficient model was duplicated by intramuscularly injecting hydrocortisone sodium succinate powder injection. After the intervention with Aconiti Lateralis Radix Praeparata, Curculiginis Rhizoma, Cinnamomi Cortex and bitter-cold herb Phellodendri Chinensis Cortex for seven days, TSH, T3, T4, 17-OHCS, COR, T, E2 of hypothalamus-pituitary-target gland axis and other relevant indexes were detected. The calmodulin expression in livers and L02 cells cultured in vitro was detected by using Western blot. RESULT: Pungent-hot herbs Aconiti Lateralis Radix Praeparata, Curculiginis Rhizoma, Cinnamomi Cortex can significantly correct indicators of hypothalamic-pituitary-gonad axis and calmodulin, whereas the bitter-cold herb Phellodendri Chinensis Cortex showed no obvious effect. CONCLUSION: The pungent-hot herb property expression may be closely related to calmodulin.

DanQi Pill protects against heart failure through the arachidonic acid metabolism pathway by attenuating different cyclooxygenases and leukotrienes B4.

BACKGROUND: Chinese herbal formulae are composed of complex components and produce comprehensive pharmacological effects. Unlike chemical drugs that have only one clear single target, the components of Chinese herbal formulae have multiple channels and targets. How to discover the pharmacological targets of Chinese herbal formulae and their underlying molecular mechanism are still under investigation. METHODS: DanQi pill (DQP), which is one of the widely prescribed traditional Chinese medicines, is applied as an example drug. In this study, we used the drug target prediction model (DrugCIPHER-CS) to examine the underlying molecular mechanism of DQP, followed by experimental validation. RESULTS: A novel therapeutic effect pattern of DQP was identified. After determining the compounds in DQP, we used DrugCIPHER-CS to predict their potential targets. These potential targets were significantly enriched in well-known cardiovascular disease-related pathways. For example, the biological processes of neuroactive ligand-receptor interaction, calcium-signaling pathway, and aminoacyl-tRNA biosynthesis were involved. A new and significant pathway, arachidonic acid (AA) metabolism, was also identified in this study. This predicted pathway alteration was validated with an animal model of heart failure (HF). Results show that DQP had effect both on thromboxane B2 (TXB2) and Prostaglandin I2 (PGI2) in different patterns. It can down-regulate the TXB2 and up-regulate the PGI2 in diverse way. Remarkably, it also had effect on cyclooxygenase (COX)-1 and COX2 by suppressing their levels, which may be the critical and novel mechanism of cardiacprotective efficacy for DQP. Furthermore, leukotrienes B4 (LTB4) receptor, another key molecule of AA metabolism which finally mediated gastrotoxic leukotrienes, was also reduced by DQP. CONCLUSIONS: The combination of drug target prediction and experimental validation provides new insights into the complicated mechanism of DQP.

Fuzi-Lizhong pill compensates hypothyroid-hypothermia via ghrelin release.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzi-Lizhong pill (FLZ) is a traditional Chinese medicine for treating patients with Spleen Yang deficient syndrome. Ghrelin, a peptide with 28 amino acid residues, plays multiple roles in thermogenesis. This study aims to explore FLZ regulating ghrelin to compensate hypothermia in rats with hypothyroid and indigestion. MATERIALS AND METHODS: In litter-matched rats, hypothermia was developed with both thyroidectomy at d1 and interscapular brown adipose (IBA) removal at d42, indigestion was induced with both high fat diet and fasting-feeding cycle from d56; the littermates with hypothermia and indigestion were administrated with FLZ from d70. Adaptive thermogenesis, thyroid hormones, metabolites, ghrelin dynamics were measured at d98. RESULTS: The results showed that plasma ghrelin levels were inversely correlated with the gastric ghrelin levels and adaptive thermogenesis in rats undergone both thyroidectomy and IBA removal. Fatty diet and FLZ enhanced the increase of plasma ghrelin of hypothyroid rats. These were supported by the changes of plasma thyroid related hormones, plasma metabolites, gastric ghrelin mRNA and protein, and the effects of fatty diet or FLZ. CONCLUSIONS: Our results suggest that more ghrelin release compensate chronic hypothermia in rats with both hypothyroidism and indigestion. It could explain the mechanisms of FLZ in relieving chronic hypothermia.

QSYQ Attenuates Oxidative Stress and Apoptosis Induced Heart Remodeling Rats through Different Subtypes of NADPH-Oxidase.

We aim to investigate the therapeutic effects of QSYQ, a drug of heart failure (HF) in clinical practice in China, on a rat heart failure (HF) model. 3 groups were divided: HF model group (LAD ligation), QSYQ group (LAD ligation and treated with QSYQ), and sham-operated group. After 4 weeks, rats were sacrificed for cardiac injury measurements. Rats with HF showed obvious histological changes including necrosis and inflammation foci, elevated ventricular remodeling markers levels(matrix metalloproteinases-2, MMP-2), deregulated ejection fraction (EF) value, increased formation of oxidative stress (Malondialdehyde, MDA), and up-regulated levels of apoptotic cells (caspase-3, p53 and tunnel) in myocardial tissue. Treatment of QSYQ improved cardiac remodeling through counter-acting those events. The improvement of QSYQ was accompanied with a restoration of NADPH oxidase 4 (NOX4) and NADPH oxidase 2 (NOX2) pathways in different patterns. Administration of QSYQ could attenuate LAD-induced HF, and AngII-NOX2-ROS-MMPs pathway seemed to be the critical potential targets for QSYQ to reduce the remodeling. Moreover, NOX4 was another key targets to inhibit the p53 and Caspase3, thus to reduce the hypertrophy and apoptosis, and eventually provide a synergetic cardiac protective effect.

[Determination of theacrine in rat plasma by RP-HPLC].

OBJECTIVE: To establish a method for the determination of theacrine in rat plasma after ig. administration of theacrine. METHOD: Blood sample was taken timely from the eyes canthus of rats. Plasma was isolated and the protein was precipitated by ethyl acetate. Then the plasma concentration of theacrine was determined with RP-HPLC. Caffeine was used as the internal standard. The chromatographic conditions were as follows: Phenomenex Luna C18 (4.6 mm x 250 mm, 5 microm) at 25 degrees C, a mixture of methanol-water (25: 75) as the mobile phase, at the flow rate of 1.0 mL x min(-1) and the detection wavelength of 290 nm. RESULT: The linear range of theacrine was 0.5-100 mg x L(-1) (R2 = 0.998 9). The lower limit of quantification was 0.5 mg x L(-1). The intra-day RSD was 1.49% 4.40% and inter-day RSD was 0.80% -10.27%. The average extraction recoveries of theacrine were 90.3% -95.8% at concentrations of 0.5, 5.0, 50 mg x L(-1). The main pharmacokinetic parameters after ig. administration of theacrine at concentration of 30 mg x kg(-1) were as follow: C(max) (35.45 +/- 30 2.68) mg x L(-1), t(max) (0.51 +/- 0.13) h, t1/2 (3.13 +/- 1.37) h, AUC(0-infinity) (2.65.39 +/- 94.71) mg x L(-1) x h. CONCLUSION: The method has been confirmed to be simple, stable, reproducible and with high specificity, and can be used for the pharmacokinetic study of theacrine in rats.

Underlying mechanism of aconitum lizhong acting on experimental hypothermia with indigestion in rats: role of ghrelin.

This study is aimed to investigate the Aconitum Lizhong pill (ALZ) pharmacological actions on hypothermia with indigestion, especially the ghrelin roles. The littermate-matched rats were randomly divided into four groups. Control did sham operation or standard diet, Model carried out interscapular brown adipose (IBA) removal with standard diet, Fat-diet did IBA removal with fat-diet, and ALZ did IBA removal and fat-diet with 4.536 g/kg/d ALZ. The potency of adaptive thermogenesis, ghrelin levels in plasma or gastric mucosa, thyroid hormones and metabolite in sera, expression of ghrelin mRNA, and protein in gastric mucous membrane were determined. ALZ relieved the hypothermia processes with indigestion, via inhibiting ghrelin expression and increasing ghrelin secretion; the dynamics from the therapy is supported with the energy changes as less body weight loss, less plasma lipid decrease, more plasma T(3) or T(4) increase with TSH decrease, and more compensation of thermogenic AUC decrease. Ghrelin played key roles in the actions of ALZ on the hypothermia with indigestion. The pharmacological mechanisms of ALZ involved the homeostasis of ghrelin expression and secretion.

[Effects of curcumin on bleomycin-induced damages in pulmonary functions in rats].

OBJECTIVE: To determine the effects of curcumin on bleomycin (BLM)-induced pulmonary fibrosis in rats. METHOD: One hundred and forty-four male Sprague-Dawley rats were randomized into 6 groups (24 rats in each group, model group, sham group, prednisone group (0.56 mg x kg(-1) x d(-1)), curcumin with low dose 5 mg group, curcumin with middle dose group 10 mg and curcumin with high dose group 20 mg per 100 g of body weight). Rats in all groups except in sham group were injected with BLM intratracheally. Curcumin with different doses were given by gavage one time everyday for 7, 14 and 28 days. Prednisone were given to rats in prednisone group, po, serving as the positive treatment group. On the 7th, 14th, 28th day, the lung functions (inspiratory resistance, maximal volutary ventilation, forced vital capacity, Fev 0.2/FVC, peak expiratory flow) were determinated in experimental rats, respectively, and the concentrations of hydroxyproline in lung homogenates of each rat were assayed. RESULT: Administration of curcumin in different doses improved lung functions of BLM-induced fibrotic rats in the all experimental days; and it decreased the concentration of hydroxyproline in lung homogenates compared with those levels in model control group; and it also lessened the hyperplasia of BLM-induced pulmonary fibrosis in rats. CONCLUSION: Administration of curcumin can suppress BLM induced pulmonary fibrosis indicated by improved respiratory function, as well as companied with low content of hydroxyproline in lung tissue of rats.

[Study on role of Chengqi Shengxue prescription in anti-tumor and immunoregulation].

OBJECTIVE: To investigate the role of Chengqi Shengxue prescription in anti-tumor and immunoregulation and to evaluate its effect on apoptosis and T lymphocyte subsets of tumor-bearing mice. METHOD: S180 ascites tumor and Lewis lung carcinoma tumor-bearing mice were used in the screening. Then 55 mice were treated randomly with the model, cyclophosphamide (30 mg x kg(-1)), or three different dosages of Chengqi Shengxue prescription (2. 4, 1.2, 0.6 g x kg(-1). After the treatment apoptosis of tumor cell and peripheral T lymphocyte subsets of tumor-bearing mice was analyzed by flow cytometry. RESULT: Lewis lung carcinoma was a nsitive tumor cell line to Chengqi Shengxue prescription. Compared with the model group, significantly increased apoptosis was observed after administration of high and medium dose of Chengqi Shengxue prescription (P < 0. 05) by PI staining. Increased early apoptosis in cancer cells was observed in all experimental doses of Chengqi Shengxue prescription by Annexin V and PI double staining (P < 0.01) . The analysis of T lymphocyte subsets showed that the percentage of CD3, CD4 and CD4/CD8 ratio decreased significantly in model group when compared with the normal ones (P < 0.01), while no change was observed in CD8. In administration groups, CD3, CD4 and CD8 were significantly lower than normal ones (P < 0.01) , but CD4/CD8 ratio did not change significantly. CONCLUSION: Chengqi Shengxue prescription has selectively inhibitive effect on the growth of mouse Lewis lung carcinoma and takes an antitransfer role. Its anti-tumor effect may be owing to inducing tumor cell apoptosis. Chengqi Shengxue prescription improves cellular immune function through enhancing CD4/CD8 ratio.