Investigation of the applicability of the zebrafish model for the evaluation of aristolochic acid-related nephrotoxicity.

BACKGROUND: The risk of compounds/drugs, including aristolochic acid-induced nephrotoxicity remains high and is a significant public health concern. Therefore, it is particularly important to select reasonable animal models for rapid screening and evaluation of different samples with complex chemical systems. The zebrafish (Danio rerio) has been used to study chemical-induced renal toxicity. However, most of the published literature was performed on individual components or drugs, and the key evidence confirming the applicability of zebrafish larvae for the evaluation of aristolochic acid-related nephrotoxicity in complex chemical systems, such as in traditional Chinese medicine (TCM), was insufficient. METHODS: High-performance liquid chromatography (HPLC) was used to determine the content of aristolochic acid (AA) in herbs and Chinese patent medicines. The zebrafish larvae at 4 days post-fertilization (dpf) were used to evaluate the nephrotoxicity of various samples, respectively, based on the phenotype of the kidney and histological, and biochemical. Transcriptome technology was used to investigate the related signaling pathways and potential mechanisms after treatment with AA, which was verified by RT-PCR technology. RESULTS: The results showed that the total amounts of AAI, AAII, and ALI ranged from 0.0004 to 0.1858 g·g-1( %) from different samples, including Aristolochia debilis, Fibraurea recisa, Asarum, Wantongjingu tablets, Jiuweiqianghuo granules, and Xiaoqinglong granules in descending order. Moreover, compared with the negative/blank control, substantial changes in phenotype, histomorphology and biochemical parameters of renal function were observed in the groups challenged with the sublethal concentration of drugs. The transcriptomics results showed the upregulation of most genes in PERK/ATF4/CHOP, ATM/Chk2/p53, Caspase/Bax/Bcl-2a, TGF/Smad/ERK, PI3K/Akt, induced by aristolochic acid analogues, which were essentially consistent with those of the q-RT-PCR experiments, highlighting the similar toxicity response to the previously published article with the other traditional evaluation model. CONCLUSION: The stability, accuracy and feasibility of the zebrafish larval model in screening and evaluating the nephrotoxicity of TCM were validated for the first time on the AAs-related drugs in a unified manner, confirming and promoting the applicability of zebrafish in assessing nephrotoxicity of samples with complex chemical character.

Jiawei Yanghe Decoction suppresses breast cancer by regulating immune responses via JAK2/STAT3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiawei Yanghe Decoction (JWYHD) is a widely used traditional Chinese medicine prescription in the clinical setting for the treatment of autoimmune diseases. Many studies showed that JWYHD has anti-tumor activities in cell and animal models. However, the anti-breast cancer effects of JWYHD and the underlying mechanisms of action remain unknown. AIM OF STUDY: This study aimed to determine the anti-breast cancer effect and reveal the underlying mechanisms of action in vivo, in vitro and in silico. MATERIALS AND METHODS: Orthotopic xenograft breast cancer mouse model and inflammatory zebrafish model were used to observe the anti-tumor effect and immune cell regulation of JWYHD. Moreover, the anti-inflammatory effect of JWYHD were evaluated by the expression of RAW 264.7 cells. JWYHD active ingredients were obtained by UPLC-MS/MS and potential targets were screened by network pharmacology. The therapeutic targets and signaling pathways predicted by computer were assessed by Western blot, real-time PCR (RT-PCR), immunohistochemistry (IHC) staining, and Enzyme-linked immunosorbent assays (ELISA) to explore the therapeutic mechanism of JWYHD against breast cancer. At last, Colivelin and Stattic were used to explore the effect of JWYHD on JAK2/STAT3 pathway. RESULTS: JWYHD significantly decreased the tumor growth in a dose-dependent manner in the orthotopic xenograft breast cancer mouse model. Flow cytometry and IHC results indicated that JWYHD decreased the expressions of M2 macrophages and Treg while increasing M1 macrophages. Meanwhile, ELISA and Western blot results showed a decrease in IL-1ß, IL-6, TNFα, PTGS2 and VEGFα in tumor tissue of JWYHD groups. The results were also verified in LPS-induced RAW264.7 cells and zebrafish inflammatory models. TUNEL assay and IHC results showed that JWYHD significantly induced apoptosis. Seventy-two major compounds in JWYHD were identified by UPLC-MS/MS and Network pharmacology. It was found that the significant binding affinity of JWYHD to TNFα, PTGS2, EGFR, STAT3, VEGFα and their expressions were inhibited by JWYHD. IHC and Western blot analysis showed that JWYHD could decrease the expression of JAK2/STAT3 pathway. Furthermore, Colivelin could reverse the decrease effect of JWYHD in vitro. CONCLUSION: JWYHD exerts a significant anti-tumor effect mainly by inhibiting inflammation, activating immune responses and inducing apoptosis via the JAK2/STAT3 signaling pathway. Our findings provide strong pharmacological evidence for the clinical application of JWYHD in the management of breast cancer.

Integrated serum pharmacochemistry, 16S rRNA sequencing and metabolomics to reveal the material basis and mechanism of Yinzhihuang granule against non-alcoholic fatty liver disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Yinzhihuang granule (YZHG) has liver protective effect and can be used for clinical treatment of non-alcoholic fatty liver disease (NAFLD), but its material basis and mechanism need to be further clarified. AIM OF THE STUDY: This study aims to reveal the material basis and mechanism of YZHG treating NAFLD. MATERIALS AND METHODS: Serum pharmacochemistry were employed to identify the components from YZHG. The potential targets of YZHG against NAFLD were predicted by system biology and then preliminarily verified by molecular docking. Furthermore, the functional mechanism of YZHG in NAFLD mice was elucidated by 16S rRNA sequencing and untargeted metabolomics. RESULTS: From YZHG, 52 compounds were identified, of which 42 were absorbed into the blood. Network pharmacology and molecular docking showed that YZHG treats NAFLD with multi-components and multi-targets. YZHG can improve the levels of blood lipids, liver enzymes, lipopolysaccharide (LPS), and inflammatory factors in NAFLD mice. YZHG can also significantly improve the diversity and richness of intestinal flora and regulate glycerophospholipid and sphingolipid metabolism. Moreover, Western Blot experiment showed that YZHG can regulate liver lipid metabolism and enhance intestinal barrier function. CONCLUSIONS: YZHG may treat NAFLD by improving the disruption of intestinal flora and enhancing the intestinal barrier. This will reduce the invasion of LPS into the liver subsequently regulate liver lipid metabolism and reduce liver inflammation.

[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.

Berberine inhibits RA-FLS cell proliferation and adhesion by regulating RAS/MAPK/FOXO/HIF-1 signal pathway in the treatment of rheumatoid arthritis.

AIMS: Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis. METHODS: Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology. RESULTS: Berberine inhibited proliferation and adhesion of RA-FLS cells, and significantly reduced the expression of MMP-1, MMP-3, RANKL, and TNF-α. Transcriptional results suggested that berberine intervention mainly regulated forkhead box O (FOXO) signal pathway, prolactin signal pathway, neurotrophic factor signal pathway, and hypoxia-inducible factor 1 (HIF-1) signal pathway. CONCLUSION: The effect of berberine on RA was related to the regulation of RAS/mitogen-activated protein kinase/FOXO/HIF-1 signal pathway in RA-FLS cells.Cite this article: Bone Joint Res 2023;12(2):91-102.

Transcriptomic profiling revealed the role of apigenin-4'-O-α-L-rhamnoside in inhibiting the activation of rheumatoid arthritis fibroblast-like synoviocytes via MAPK signaling pathway.

BACKGROUND: Activated fibroblast-like synoviocyte (FLS) played a significant role in the pathogenesis and progression of rheumatoid arthritis (RA). Apigenin-4'-O-α-L-rhamnoside showed remarkable effects against RA, however, no relevant studies on pharmacology of apigenin-4'-O-α-L-rhamnoside yet, the effects and underlying molecular mechanism of apigenin-4'-O-α-L-rhamnoside on RA are still unclear. PURPOSE: This study aimed to investigate the therapeutic effects and mechanisms of apigenin-4'-O-α-L-rhamnoside on RA-FLS cells by transcriptomic analysis. METHODS: In vitro, RA-FLS cell viability and migration were measured by CCK-8 and scratch assays, respectively. The effects of apigenin-4'-O-α-L-rhamnoside on inflammatory levels of MMP-1, MMP-3, RANKL and TNF-α in RA-FLS cells were detected using ELISA kits. High-throughput transcriptome analysis was performed to screen the key genes and related pathways of apigenin-4'-O-α-L-rhamnoside inhibit RA-FLSs, and the result of which were validated by RT-qPCR and western blot. Furthermore, in vivo, we also evaluated the effects of apigenin-4'-O-α-L-rhamnoside in rat with CIA. RESULTS: Apigenin-4'-O-α-L-rhamnoside significantly suppressed RA-FLS migration, exerted remarkable inhibiting effects on the expression levels on MMP-1, MMP3, RANKL and TNF-α in RA-FLS cells. It seemed that MAPK signaling pathway might be closely related to the pathogenesis of RA by down-regulated relevant core targets (MAPK1, HRAS, ATF-2, p38 and JNK). Moreover, apigenin-4'-O-α-L-rhamnoside attenuated the severity of arthritis in CIA rat. CONCLUSION: Apigenin-4'-O-α-L-rhamnoside inhibited pro-inflammatory cytokine, chemokine and MMPs factors production of RA-FLS by targeting the MAPK signaling pathway, which provided a scientific basis for potential application in the treatment of RA.

[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.

Lipidomics Indicates the Hepatotoxicity Effects of EtOAc Extract of Rhizoma Paridis.

Rhizoma Paridis is a traditional Chinese medicine commonly used in the clinical treatment of gynecological diseases. Previous studies have shown that aqueous extracts of Rhizoma Paridis exhibit some hepatotoxicity to hepatocytes. Here, using lipidomics analysis, we investigated the potential hepatotoxicity of Rhizoma Paridis and its possible mechanism. The hepatic damaging of different solvent extracts of Rhizoma Paridis on zebrafish larvae were determined by a combination of mortality dose, biochemical, morphological, and functional tests. We found that ethyl acetate extracts (AcOEtE) were the most toxic fraction. Notably, lipidomic responsible for the pharmacological effects of AcOEtE were investigated by Q-Exactive HF-X mass spectrometer (Thermo Scientific high-resolution) coupled in tandem with a UHPLC system. Approximately 1958 unique spectral features were detected, of which 325 were identified as unique lipid species. Among these lipid species, phosphatidylethanolamine cardiolipin Ceramide (Cer), lysophosphatidylinositol sphingosine (Sph), etc., were significantly upregulated in the treated group. Pathway analysis indicates that Rhizoma Paridis may cause liver damage via interfering with the glycerophospholipid metabolism. Collectively, this study has revealed previously uncharacterized lipid metabolic disorder involving lipid synthesis, metabolism, and transport that functionally determines hepatic fibrosis procession.

Hepatotoxicity induced by PPâ ¥ and PPâ ¦ in zebrafish were related to the Cholesterol disorder.

BACKGROUND: PPⅥ2 and PPⅦ3 were a group of Pennogenin compounds extracted from the Paris polyphylla and caused hepatotoxicity in human, while the potential underlying mechanism was unclear. PURPOSE: To evaluated the adverse effects of PPⅥ and PPⅦ on the liver in the zebrafish. METHOD: In this study, 4dpf zebrafish were used for acute toxicity test, LC0 was calculated, and 1/2LC0 and 3/5LC0 were selected for pathological section and liver area measurement to verify the hepatotoxicity of PPⅥ and PPⅦ. Etabonomics study was then conducted to further explore the mechanism of hepatotoxicity of PPⅥ and PPⅦ. Lovastatin was used as an inhibitor, and PCR was used to verify the results. RESULT: The result showed that under the condition of sub-lethal concentration exposure, hepatotoxicity-included changes in liver phenotype (liver area), hepatocyte swelling and degeneration, liver cell apoptosis and disturbed biochemical index were observed in zebrafish treated with PPⅥ and PPⅦ. Furthermore, the transcriptome was conducted to confirm the toxicity mechanism shared with PPⅥ and PPⅦ, and we found that steroid biosynthesis process and the related target genes were mainly affected. While, lovastatin treatment effectively ameliorated PPⅦ-induced zebrafish liver injury by improving the liver tissue structure and regulate the expression of associated genes including HMGCRA, SREBP, LSS, CYP2R1, PIK3R3A, GDPD1 and PFKFB-2. CONCLUSION: This study was the first investigation to provide the direct evidence of hepatotoxicity of PPⅥ and PPⅦ in vivo zebrafish model, which were related to the steroid biosynthesis. furthermore, in lovastatin played an important role in protection against hepatotoxicity induced by PPVI and PPⅦ by regulating the cholesterol metabolism.

Combined Metabolomics and Network Toxicology to Explore the Molecular Mechanism of Phytolacca acinose Roxb-Induced Hepatotoxicity in Zebrafish Larvae in Vivo.

Phytolacca acinosa Roxb (PAR), a traditional Chinese medicine, has been widely used as a diuretic drug for a long period of time for the treatment edema, swelling, and sores. However, it has been reported that PAR might induce hepatotoxicity, while the mechanisms of its toxic effect are still unclear. In this study, network toxicology and metabolomic technique were applied to explore PAR-induced hepatotoxicity on zebrafish larvae. We evaluated the effect of PAR on the ultrastructure and the function of the liver, predictive targets, and pathways in network toxicology, apoptosis of liver cells by PCR and western blot, and metabolic profile by GC-MS. PAR causes liver injury, abnormal liver function, and apoptosis in zebrafish. The level of arachidonic acid in endogenous metabolites treated with PAR was significantly increased, leading to oxidative stress in vivo. Excessive ROS further activated the p53 signal pathway and caspase family, which were obtained from KEGG enrichment analysis of network toxicology. The gene levels of caspase-3, caspase-8, and caspase-9 were significantly increased by RT-PCR, and the level of Caps3 protein was also significantly up-regulated through western blot. PAR exposure results in the liver function abnormal amino acid metabolism disturbance and motivates hepatocyte apoptosis, furthermore leading to liver injury.

[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.