[Hepatotoxicity and mechanism of Rhododendri Mollis Flos based on zebrafish model].

This study used the zebrafish model to explore the hepatotoxicity of Rhododendri Mollis Flos(RMF). The mortality was calculated according to the number of the survival of zebrafish larvae 4 days after fertilization under different concentration of RMF, and the dose-toxicity curve was fitted to preliminarily evaluate the toxicity of RMF. The liver phenotypes under the sublethal concentration of RMF in the treatment group and the blank control group were observed by hematoxylin-eosin(HE) staining and acridine orange(AO) staining. Meanwhile, the activities of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were determined to confirm the hepatotoxicity of RMF. Real-time quantitative polymerase chain reaction(real-time PCR) and Western blot were used to determine the expressions of genes and proteins in zebrafish larvae. Gas chromatography time-of-flight mass spectrometry(GC-TOF-MS) was used to conduct untargeted metabolomics testing to explore the mechanism. The results showed that the toxicity of RMF to zebrafish larvae was dose-dependent, with 1 100 µg·mL~(-1) of the absolute lethal concentration and 448 µg·mL~(-1) of sublethal concentration. The hepatocyte apoptosis and degeneration appeared in the zebrafish larvae under the sublethal concentration of RMF. The content of ALT and AST in zebrafish larvae at the end of the experiment was significantly increased in a dose-dependent manner. Under the sublethal concentration, the expressions of genes and proteins related to apoptosis in zebrafish larvae were significantly increased as compared with the blank control group. The results of untargeted metabolomics showed that the important metabolites related to the he-patotoxicity of RMF were mainly enriched in alanine, aspartic acid, glutamic acid, and other pathways. In conclusion, it is inferred that RMF has certain hepatotoxicity to zebrafish larvae, and its mechanism may be related to apoptosis.

[Anti-tumor and analgesic activity evaluation and mechanism of Compound Kushen Injection].

This study aims to evaluate the anti-tumor and analgesic activities of Compound Kushen Injection(CKI) based on zebrafish model in vivo and investigate the anti-tumor mechanism. To be specific, zebrafish tumor xenotransplantation model was established by microinjection of murine LPC H12 cells into yolk sac. Then the high-dose CKI(H-CKI), medium-dose CKI(M-CKI), low-dose CKI(L-CKI) groups, and the model group were set. The anti-tumor activity of CKI was evaluated with the tumor area growth fold and integral absorbance(IA) growth fold 72 h after administration. The peripheral pain and central pain in zebrafish were respectively induced with acetic acid(AA) and phorbol myristate acetate(PMA). Zebralab ViewPoint system was employed to monitor behavioral trajectory of zebrafish, and movement times, movement time, movement distance, and movement velocity were used to evaluate the analgesic activity of CKI. Finally, real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression levels of apoptosis-related B lymphocyte tumor-2(Bcl-2) and phosphatidylinositol-3-kinase(PI3 K)/protein kinase B(Akt or PKB) pathway-related genes, for the verification of the anti-tumor mechanism. Compared with the model group, M-CKI and H-CKI significantly reduced the growth folds of tumor area and IA, relief the peripheral pain and central pain. The mechanism was that CKI can up-regulate the expression of cysteine aspartic acid specific protease-3(caspase-3, Casp3) and caspase-9(Casp9), down-regulate the expression of phosphoinositide 3-kinase(PI3 K) and Akt, and significantly reduce the expression of Bcl-2, hypoxia-inducible factor-1α(HIF-1α), and vascular endothelial growth factor(VEGF). In conclusion, CKI has significant inhibitory effect on tumor growth and pain, which is related to the PI3 K/Akt signaling pathway. The pathway mediates cell apoptosis, suppresses tumor growth, and alleviates tumor pain.

[Serum metabolic profile involving protective effect of "Neiguan"(PC6)-electroacupuncture preconditioning in rats with myocardial ischemia reperfusion injury].

OBJECTIVE: To observe the effect of "Neiguan" (PC6)-electroacupuncture (EA) preconditioning on serum metabolites in myocardial ischemia-reperfusion injury (MIRI) rats, so as to reveal its mechanism underlying improvement of ischemic myocardium from metabonomics. METHODS: A total of 48 male SD rats were randomly divided into control, model, EA "Neiguan"(PC6) and EA "Hegu"(LI4) groups (n＝12 rats/ group). Rats of the control group were just banded on animal boards for 30 min, once daily for 7 days. The MIRI model was established by occlusion of the left anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 1 h, and rats of the model group were also banded as those in the control group. Before modeling, EA (10 Hz/50 Hz, 1 mA) was applied to bilateral "Neiguan"(PC6) and "Hegu"(LI4) for 30 min, once daily for 7 successive days. After the treatment, serum samples were collected to be analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy. The orthogonal partial least squares discriminate analysis (PLS-DA) was employed to distinguish the serum differential metabolic profile of rats in different groups and identify potential biomarkers. RESULTS: After modeling, the ECG of model group and electroacupuncture groups showed T wave towering, and there was no obvious ST segment between R wave and T wave. The T wave decreased more than 0.2 mV after reperfusion, and there was no obvious ST segment. Compared with the control group, MIRI induced significant changes of metabolites in the serum including increase of acetoacetate acid, lectic acid, creatine, glycerol and glucose, and decrease of alanine, glutamine, glycerophosphoryl choline and phosphorylcholine. In comparison with the model group, PC6-EA preconditioning induced significant changes, including an increase of glucose, and a decrease of leucine，isoleucine, valine，3-hydroxybutyric acid，lactate，acetate，acetone，acetoacetate acid，pyruvic acid，glutamine，creatine and glycerol. There is no significant difference in metabolic patterns between "Hegu" group and model group. Metabolic pathway enrichment analysis indicated that the protective effect of PC6-EA pretreatment was realized mainly by regulating pathways of glycolysis, gluconeogenesis, citric acid metabolism, pyruvate metabolism, ketone body metabolism, etc. CONCLUSION: PC6-EA pretreatment has a role in regulating gluconeogenesis, pyruvate metabolism, amino metabolism, ketone body metabolism and energy metabolism in rats with MIRI, which maybe contribute to its protective effect on ischemic myocardium, but the specific metabolic pathways and mechanisms need being studied further.

[Electroacupuncture and moxibustion pretreatments reduce cardiomyocyte apoptosis and autophagy in rats with myocardial ischemia/reperfusion injury].

OBJECTIVE: To observe the effect of "Neiguan" (PC6)-electroacupuncture (EA) or moxibustion (Moxi) pretreatment on myocardial apoptosis and expression of autophagy related proteins light chain (LC) 3-Ⅰ and LC3-Ⅱ in rats with myocardial ischemia/reperfusion injury (MIRI), so as to explore their mechanisms underlying improvement of MIRI. METHODS: Forty SD rats (half male and half female) were randomly divided into sham operation, model, ischemic preconditioning (IP), EA and Moxi groups (n＝8 in each group). EA (10 Hz/50 Hz, 1 mA) or Moxi (ignited moxa stick) was respectively applied to bilateral "Neiguan" (PC6) for 20 min, once daily for 7 days. The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 60 min. The ultrastructural changes and autophagy of myocardial cells were observed by electron microscopy (EM), and the myocardial cellular apoptosis [apoptotic index ＝ (number of apoptotic cells/total number of cardiomyocytes)×100%] was detected by the terminal deoxyribonucleotidyl transferase mediated dUTP nick end labelling (TUNEL) method. The expressions of LC3-Ⅰ and LC3-Ⅱ proteins (markers for autophagy) in myocardial tissue were detected by Western blot. RESULTS: Following MI, EM observation revealed a vague structure of cardiomyocytes and muscular horizontal grain, dissolution of myofibers, mitochondrial swelling, some autophagic vacuoles and autophagic lysosomes at different degrees and surrounded by a double membrane in the model group, these situations were apparently milder in the EA and Moxi groups. The apoptosis index, myocardial LC3-Ⅰ and LC3-Ⅱ protein expression levels, and the ratio of LC3-Ⅱ/Ⅰ were significantly increased in the model group relevant to the sham operation group (P<0.05). After the treatment, the apoptosis index, the expression level of myocardial LC3-Ⅱ protein and the ratio of LC3-Ⅱ/Ⅰ were considerably down-regulated in the IP, EA and Moxi groups in comparison with those in the model group (P<0.05). The effect of EA was obviously superior to those of IP and Moxi in down-regulating the apoptosis index (P<0.05), but obviously inferior to those of IP and Moxi in down-regulating the levels of LC3-Ⅱ and LC3-Ⅱ/Ⅰ (P<0.05). No significant changes were found in the expression of LC3-Ⅰ after IP, EA and Moxi interventions in comparison with the model group (P>0.05), and no significant differences were observed in the apoptosis index and levels of LC3-Ⅱ and LC3-Ⅱ/Ⅰ between the IP and Moxi groups (P>0.05)．. CONCLUSION: Both EA and moxibustion pretreatments, similar to IP, have a positive role in reducing myocardiocyte apoptosis and regulating autophagy-related protein expression in MIRI rats, which maybe contribute to their protective effects on ischemic myocardium.

[Effect of Electroacupuncture at "Neiguan"(PC 6) on Serum and Myocardial Metabolites in Rats with Myocardial Ischemia Reperfusion Injury Based on Nuclear Magnetic Resonance Spectroscopy].

OBJECTIVE: We have repeatedly demonstrated that electroacupuncture (EA) of "Neiguan"(PC 6) can improve myocardial ischemia in rats. The present study was designed to investigate the metabolomic profile of peripheral blood se-rum and myocardium involving EA-induced improvement of myocardial ischemia-reperfusion injury (MIRI) in rats by using nuclear magnetic resonance spectroscopy. METHODS: Thirty male SD rats were equally randomized into blank control, model and EA groups. Rats of the control group were only banded for 20 min, once a day for 7 days. The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 60 min, and rats of the model group were banded as those in the control group. EA (10 Hz/50 Hz, 1 mA) was applied to bilateral PC 6 for 20 min, once daily for 7 days. The blood samples and left ventricular myocardial tissues were collected for assaying the profiles of differential metabolites using 1H nuclear magnetic resonance (1H NMR) spectroscopy and multivariate statistical analysis such as the principal components analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) with SIMCA-P software 12.0. RESULTS: A total of 19 differential metabolites (17 down-regulated, 2 up-regulated) in the serum and 14 differential metabolites (13 down-regulated and 1 up-regulated) in the ischemic left myocardium were identified after MIRI. Of the 19 serum differential metabolites, amino acids (leucine, isoleucine, valine,alanine, lysine, glycine, glutamine), 3-hydroxy butyric acid (3-HB), lactic acid, acetate, N-acetyl glycoprotein (NAc), acetone, acetoacetate, succinate, polyunsaturated fatty acids (PUFA), creatine, glycerophosphocholine (GPC) were down-regulated; while low density lipoprotein (LDL), LDL/very low density lipoprotein(LDL/VLDL)and glucose obviously up-regulated. Of the 14 myocardial differential metabolites, amino acids (alanine, lysine, glutamate, glutamine, aspartate, taurine, glycine, threonine), GPC, creatine, lactic acid, adenosine monophosphate (AMP), nicotinamide adenine dinucleotide (NAD＋) were significantly decreased, and glucose was up-regulated. Following EA treatment, most of the decreased serum differential metabolites except acetone, acetoacetate and PUFA, and the increased serum LDL, LDL/VLDL and glucose recovered, basically close to the control level; and the decreased myocardial creatine, GPC and NAD＋ were also apparently up-regulated and the increased myocardial glucose was down-regulated. But, myocardial threonine and AMP still presented a decreasing state. Although the pattern of myocardial differential metabolites of the EA group had a trend to be close to the control group, the significant difference still existed, while the metabolic pattern of serum metabolites in the EA group was close to that of the control group. CONCLUSION: EA stimulation of PC 6 can regulate serum or/and myocardial metabolites as amino acids, carbohydrates, lipids, etc. in MIRI rats, of which both serum and myocardial creatine, GPC and glucose may be jointly confer a favorable potential for EA-induced improvement of MIRI.

[Effect of Electroacupuncture and Moxibustion Pretreatment on Expression of Autophagy Related Proteins LC 3 and Beclin 1 in Rats with Myocardial Ischemia-reperfusion Injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) and moxibustion (Moxi) pretreatment on myocardial pathological and structural changes and expression of autophagy related protein LC 3 Ⅰ/Ⅱ and Beclin 1 in rats with myocardial ischemia-reperfusion injury (MI/RI), so as to explore their mechanisms underlying improving MI/RI. METHODS: Forty SD rats were randomly divided into sham operation, model, ischemic preconditioning (IP), EA and Moxi groups (n＝8 in each group). EA (10 Hz/50 Hz，1 mA) or Moxi (ignited moxa stick) was respectively applied to bilateral "Neiguan"(PC 6) for 20 min, once daily for 7 days. The MI/RI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 60 min. The left ventricular (LV) tissue samples were collected and analyzed for pathological (H.E. staining) and ultrastructural changes, for myocardial apoptosis (apoptotic index＝ number of apoptotic cells/total number of cardiomyocytes×100%) with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, and for the expression of LC 3 and Beclin 1 in myocardial cells with Western blot. RESULTS: Following MI/RI, H．E. staining revealed a disorder of arrangement of cardiomyocytes with vague border, inflammatory cell infiltration, intracellular swelling with bleeding, necrosis and dissolution of partial striated muscles of the left ventricle under light microscope, and dual staining of Uranyl acetate and leadnitrate showed atrophy, arrangement disorder, dissolution, necrosis, and interstitial edema of partial myocardial fibers, mitochondrial structural disorder, vacolation, and large body of autophagosomes with bilayers, etc. in ultrastructure, which was relatively lighter in both EA and Moxi groups. The apoptosis index, expression levels of myocardial LC 3 Ⅱ and Beclin 1 and the ratio of LC 3 Ⅱ/LC 3 Ⅰ were significantly higher in the model group than those in the sham operation group (P<0.01), but the expression level of LC 3 Ⅰ was considerably down-regulated in the model group relevant to the sham operation group (P<0.01). Following the intervention and MI preconditioning, the increased apoptosis index and expression levels of LC 3Ⅱ and Beclin 1 proteins and the ratio of LC 3Ⅱ/LC 3 Ⅰ were obviously down-regulated in the IP, EA and Moxi groups relevant to the model group (P<0.01), and the decreased expression of LC 3 Ⅰ protein was up-regulated obviously in the 3 treatment groups (P<0.05，P<0.01). The effects of EA were significantly superior to those of IP and Moxi groups in down-regulating apoptosis index and expression of LC 3 Ⅱ and Beclin 1 and the ratio of LC 3 Ⅱ/LC 3 Ⅰ and in up-regulating expression of LC 3 Ⅰ (P<0.05, P<0.01). CONCLUSION: Both EA and Moxi preconditioning of PC 6 have a protective effect on ischemic myocardium in MI/RI rats, which is probably related to their effects in regulating expression of myocardial autophagy proteins as LC 3 Ⅰ/Ⅱ and Beclin 1.