Electroacupuncture Inhibits Atherosclerosis through Regulating Intestinal Flora and Host Metabolites in Rabbit.

METHODS: In this study, general rabbit conditions, vascular histology, metabolites, and intestinal flora structures were analyzed. Integrated analysis of metabolomics and 16S rRNA sequencing were performed. All the rabbits were randomly divided into four groups. The rabbit model of atherosclerosis was established. The histopathological change in the common carotid artery was assessed by HE staining and the structural change in the flora by 16S rRNA sequencing. HPLC-TOF-MS and Agilent MPP 12.1 were integrated to identify and screen out differential metabolites. Correlational analyses of every differential metabolite with intestinal flora were integrated on Omicshare platform. RESULTS: Atherosclerotic rabbits showed obvious changes in general conditions, significant fibrous cap and necrotic center on carotid artery, abnormal intestinal bacteria structure, and metabolites levels. Electroacupuncture improved the conditions, reduced lipid deposition on the carotid artery wall, diversified intestinal flora, and normalized host metabolism. Integrated analysis showed that 149 altered metabolites were related to 22 intestinal flora, among which eight intestinal floras and 21 metabolites have relationships with atherosclerosis. CONCLUSION: Electroacupuncture can effectively reverse atherosclerosis through manipulating the structural feature of intestinal flora to influence the host metabolites. The possible mechanisms involved activating signal pathways through host metabolites or affecting the activity of cardiovascular-related enzymes, or regulating host lipid metabolism directly.

[Systematic review on the animal experiments of electroacupuncture for hyperlipidemia combined with atherosclerosis].

OBJECTIVE: To systematically review the effectiveness and safety of electroacupuncture (EA) on hyperlipidemia combined with atherosclerosis in animal experiments. METHODS: The relevant animal experiments of EA treatment for hyperlipidemia combined with atherosclerosis were retrieved from the CNKI, WANFANG, VIP, CBM, PubMed, Embase and the Cochrane Library up to March 19, 2018 since the establishment of databases. Two researchers in the project team screened the literature and extracted the information such as experiment objects, experiment steps and experiment results separately. The literature assessment was evaluated. Meta analysis was performed with RevMan 5.3 software. RESULTS: Eventually, 29 eligible experimental researches were included and the average score in the quality assessment was 5.1, at the moderate level. Meta analysis showed that compared with the model control group, after EA treatment, the levels of the total cholesterol (TC) [SMD -2.29, 95% CI (-2.94, -1.64), P<0.000 01], triglyceride (TG) [SMD -1.30, 95% CI (-1.76, -0.84), P<0.000 01] and low density lipoprotein-cholesterol (LDL-C) [SMD -2.64, 95% CI (-3.29, -1.99), P<0.000 01] were significantly reduced and the level of high density lipoprotein-cholesterol (HDL-C) [SMD 0.55, 95% CI (0.06, 1.04), P =0.03] was increased, indicating the significant differences. CONCLUSION: On the base of the current limited evidences, EA significantly reduces the levels of TC, TG and LDL-C and slightly increases the level of HDL-C in the animal models of hyperlipidemia combined with atherosclerosis.

[Effects of electroacupuncture on CYP7A1 expression in liver of rabbits with atherosclerosis].

OBJECTIVE: To observe the effects of electroacupuncture (EA) at "Neiguan" (PC 6), "Guanyuan" (CV 4) and "Zusanli" (ST 36) on CYP7A1 expression in liver of rabbits with atherosclerosis (AS), and to explore the mechanism of acupuncture for prevention and treatment of atherosclerosis. METHODS: A total of 26 male rabbits were adaptively fed for 1 week in different cages. Seven rabbits were randomly divided into a blank group, and the remaining 19 rabbits were divided into a model group. The blank group was fed with normal diet, while the model group was fed with high-fat diet. After high-fat diet for 4 weeks, the rabbits in the model group were treated with balloon injury surgery of the common carotid artery; after surgery, the rabbits were fed with high-fat diet for 4 weeks. One rabbit was randomly selected from the blank group and model group to obtain the pathological section of carotid artery; the HE staining was used to observe the pathomorphology of atheromatous plaque to determine the success of modeling or not. After successful establishment of modeling, 18 rabbits were randomly divided into a AS model group, an EA group and a medication group, 6 rabbits in each one. The rabbits in the AS model group received no treatment; the rabbits in the medication group were treated with intragastric administration of atorvastatin calcium tablets; the rabbits in the EA group were treated with EA at "Neiguan" (PC 6), "Guanyuan" (CV 4) and "Zusanli" (ST 36), once a day, 20 min per treatment; six-day EA treatment constituted one course, and totally 4 courses were given with an interval of 1 day between courses. After treatment, vein blood was collected from rabbit ear, and cholesterol (CHO), triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured in each group; the CYP7A1 protein expression in rabbit liver was measured by Western blot method, and CYP7A1 mRNA expression in rabbit liver was measured by RT-PCR. RESULTS: Compared with the blank group, the contents of CHO, TG and LDL in the AS model group were significantly increased, but HDL was significantly decreased, and the expression of CYP7A1 protein and CYP7A1 mRNA in the liver were significantly decreased (all P<0.01). After treatment, compared with the model group, the contents of CHO, TG and LDL in the EA group and medication group were significantly reduced, but HDL was significantly increased, and the expression of CYP7A1 protein and CYP7A1 mRNA in the liver were significantly increased (all P<0.01). The significant difference of each outcome between the EA group and medication group was not observed (all P>0.05). CONCLUSION: EA could significantly improve blood lipid and promote the expression of CYP7A1 mRNA in rabbits with atherosclerotic, which may be one of the mechanisms of EA for atherosclerosis.

[Effect of Electroacupuncture on Expression of Scavenger Receptor A I in Peritoneal Macrophages of Atherosclerotic Rabbits].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of scavenger receptor A I (SR-A I) protein and mRNA in peritoneal macrophages of atherosclerosis (AS) rabbits. METHODS: A total of 26 New Zealand rabbits were used in the present study, among them, 7 were randomly selected to constitute a blank control group, and the rest 19 rabbits were fed with high-fat diet combined with carotid artery balloon injury for establishing AS model. Eight weeks after high-fat forage feeding, one high-fat fed rabbit and one normal rabbit were randomly selected for verifying the success of modeling. HE staining was employed to examine pathological changes of the common carotid artery after paraffin section. Then the rest 18 rabbits were randomly divided into model, EA and medication groups (n=6 rabbits in each). EA (2 Hz, 1 mA) was applied at "Guanyuan"(ST 25)and bilateral "Zusanli"(ST 36) and "Neiguan" (PC 6) for 20 min. The rabbits of the medication group were treated by gavage of Atorvastatin Calcium (1 mg•kg-1• d-1), and those of the model group treated by gavage of distilled water (2 mL•kg-1•d-1). The treatment was given once daily for 4 weeks, with one day's interval between every two weeks. Three days before termination of the experiments, sterile starch solution (3-4 mL/animal) was injected into the peritoneal cavity, and at the end of the experiment, the peritoneal fluid was collected and centrifugated to be cultivated in Dulbecco's Modified Eagle Medium (DMEM) for collecting the macrophages. The expression of SR-A I protein and mRNA in macrophages was assayed by Western blot and PCR, respectively. RESULTS: HE staining displayed an injury of the inner membrane of the carotid artery marked by observable atherosclerotic plaques, interruption, thickening and uplift, inflammatory cell infiltration, etc. in the AS model rabbits which were relatively milder in both EA and medication groups. The expression levels of SR-A I protein and mRNA were significantly up-regulated in the model group relevant to the blank control group (P<0.01), and considerably down-regulated in both EA and medication groups compared with the model group (P<0.01). There were no statistical differences between the EA and medication groups in the decreased levels of expression of SR-A I protein and mRNA (P>0.05). CONCLUSION: EA intervention may improve the severity of atherosclerosis to a certain degree in AS rabbits, which is possibly associated with its effect in inhibiting the expression of SR-A I protein and mRNA in the peritoneal macrophages.

[Effects of Electroacupuncture on Expression of Myocardial Chloride Channel-2 and CLCA Proteins in Mice with Acute Myocardial Ischemia].

OBJECTIVE: To observe the influence of electroacupuncture stimulation (EA) of "Neiguan" (PC 6)/"Lieque" (LU 7) on ST segment of electrocardiogram (ECG) and the expression of myocardiac chloride channel (CLC)-2 and calcium (Ca2+)-activated chloride channel accessory (CLCA) proteins in acute myocardial ischemia (AMI) mice, so as to explore its mechanisms underlying improvement of AMI. METHODS: Thirty ASIC 3-/- knock out mice were randomly divided into control, AMI model, EA-Neiguan (PC 6), EA-Lieque (LU 7) and EA-non-acupoint groups. The AMI model was induced by multiple subcuta-neous injection of isoprenaline (ISO, 0.5 g/L, 20 mg/kg). EA was applied to bilateral PC 6, LU 7, or non-acupoint[the mid-point between "Tianshu" (ST 25) and "Shenque" (CV 8)] for 20 min, once daily for 7 days. The ST segment of ECG of the standard Ⅱlimb-lead was recorded using a PowerLab data acquisition device, the change of myocardial histology observed by using microscope after HE staining, the activity of serum superoxide dismutase (SOD) detected using Colorimetric method, and the expression of CLC-2 and CLCA proteins of the left ventricle myocardium detected by Western blot. RESULTS: Outcomes of HE staining showed that the ischemic injury (sarcoplasm swelling and necrosis, etc.) of the left ventrical myocardial tissue after mo-deling was relatively milder in the EA-PC 6 group, not in the EA-non-acupoint group. The SOD activity was significantly lower in the model group than in the control group (P<0.01), and obviously increased in the EA-PC 6 group(P<0.01), but not in the EA-non-acupoint group (P>0.05). The expression levels of myocardial CLC-2 and CLCA proteins were significantly up-regulated in the model group compared with the control group (P<0.01) and markedly down-regulated in the EA-PC 6 group (not in the EA-non-acupoint group) in comparison with the model group(P<0.01), suggesting a specificity of effects of EA-PC 6 in improving myocardial injury and down-regulating CLC-2 and CLCA protein expression. CONCLUSIONS: EA of "Neiguan" (PC 6) can effectively suppress the increase of expression of CLC-2 and CLCA proteins in the left ventricle myocardium, which may contribute to its effect in relieving myocardial injury in mice.

Effects of acupuncture on the tissue distribution of Paclitaxel in lung carcinoma mice.

OBJECTIVE: To study whether acupuncture affects the tissue distribution of Paclitaxel in mouse lung carcinoma. METHODS: Totally 90 mice were divided into Paclitaxel group, Paclitaxel + Feishu (BL13) group, and Paclitaxel + Lingtai (DU10) group. Each group was consisted of 30 mice. After Paclitaxel injection, the mice received electro-acupuncture at Feishu or Lingtai acupoints once a day for 8 days. The effect of acupuncture on the tissue distribution of Paclitaxel was evaluated using high-performance liquid chromatography at 1, 2, and 3 h, respectively. The lung, liver, spleen, and kidney were examined for the concentration of Paclitaxel seperately. RESULTS: Paclitaxel was widely distributed in various organs, particularly in the lung, liver, and kidney. Acupuncture at Lingtai or Feishu acupoints resulted in an obvious decrease of Paclitaxel distribution in kidney and delayed Paclitaxel distribution in liver. Meanwhile, it increased the time of metabolism. Acupuncture at Feishu acupoint facilitated the delivery of Paclitaxel to lung more effectively than did acupuncture at Lingtai acupoint. CONCLUSIONS: Applying acupuncture at particular acupoints can influence tissue distribution of Paclitaxel. Tissue distribution change might be one of the mechanisms of acupuncture treatment during chemotherapy.

[Effects of acupuncture on distribution taxis of paclitaxel in mice with lung cancer].

OBJECTIVE: To observe the effects of acupuncture at "Feishu" (BL 13) and "Lingtai" (GV 10) on distribution taxis of paclitaxel in mice with lung cancer to discuss targeted relationship between acupoints and corresponding viscera. METHODS: According to randomized digital table, 315 SPF-grade BALB/C female mice were divided into 7 groups: blank group (group A), model group (group B), medication group (group C), acupuncture at non-acupoint group (group D), acupuncture at Feishu group (group E), acupuncture at Lingtai group (group F) and acupuncture at Feishu and Lingtai group (group G), 45 mice in each one. Except the blank group, the remaining groups were treated with N-nitroso-tris-chloroethyl urea (NTCU) to establish the model of squamous-cell carcinoma. After model establishment, group A, group B and group C were not treated with acupuncture; group A and group B were treated with intraperitoneal injection of 0.9% sodium chlorvde solution by 6 mL/kg while group C was treated with intraperitoneal injection of paclitaxel by 6 mL/kg. The group D, group E, group F and group G were treated with acupuncture at non-acupoint, "Feishu" (BL 13), "Lingtai" (GV 10) and "Feishu" (BL 13) plus "Lingtai" (GV 10), respectively, then were intraperitoneally injected with paclitaxel by 6 mL/kg. The treatment was all given once a day for continuous 10 days. 15 min, 30 min, 1 h, 2 h, 8 h, 12 h and 24 h after the treatments, 6 mice in each group were randomly selected and sacrificed to collect samples of lung, liver, spleen, kidney and heart, etc. High performance liquid chromatography was applied to measure the concentration of paclitaxel in each organ (lung, liver, spleen, kidney and heart) at different time points. RESULTS: (1) The content of paclitaxel in lung, kidney and heart reached the peak at 2 h, then decreased significantly in group C, group D, group E, group F and group G; the content of paclitaxel in spleen showed downtrend at each time point. The content of paclitaxel in liver reached the peak at 2 h in group C and group D; the content of paclitaxel reached the peak at 8 h in group E, group F and group G. (2) The content of paclitaxel in lung in group E and group G was higher than that in group C and group D at each time point (all P < 0.01); the content of paclitaxel in lung in group F was higher than that in group C (P < 0.01) and group D (P < 0.01) only at time point of 2 h. The content of paclitaxel in lung in group G was higher than that in group F at each time point (all P < 0.05). There was no statistical difference between group G and group E (all P > 0.05). CONCLUSION: Acupuncture at "Feishu" (BL 13) and "Lingtai" (GV 10) could influ- ence the metabolism of paclitaxel in lung-cancer mice, leading to distribution change in each organ. As a result, it could cause targeting effects, which is more significant at "Feishu" (BL 13) and "Lingtai" (GV 10).

The impacts of along-channel acupuncture on the protein expressions of the chloride channel of the rats with myocardial ischemia.

Recent evidence suggests that chloride (CL(-)) channels are involved in myocardial ischemia. In this study, the impact of acupuncture on the protein expressions of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and CLC-2 CL(-) channel of the rats with myocardial ischemia were tested and its mechanism was explored. The rats for experiment were distributed randomly into 5 groups: blank control group, modeling control group, Neiguan (PC-6) treatment group, Lieque (LU-7) control group, and Non-acupoint control group. The rats of all groups, except the blank control group, had myocardial ischemia via multiple subcutaneous injection of isoproterenol (ISO). Electroacupuncture treatment was given to Neiguan (PC-6) treatment group, Lieque (LU-7) control group, and Non-acupoints control group, respectively, once a day for 7 days. The results show that acupuncture can alleviate the myocardial ischemia of cardiac tissue, decrease significantly the activities of serum SOD and MDA, and thereby influence the protein expressions of CFTR and CLC-2 in CL(-) channels. The results of the study implies that acupuncture suppresses the pathological changes of cardiac tissue of rats with myocardial ischemia and regulates the protein expression of CFTR and CLC-2 CL(-) channels, which may serve as one possible mechanism to reduce myocardial ischemia.

Effects of acupuncture at Taixi acupoint (KI3) on kidney proteome.

This research explored and identified the protein composition of rat kidneys after acupuncture at the Taixi acupoint (KI3). Twelve adult male Wistar rats were randomly divided into a control group (n = 6) and an acupuncture group (n = 6). Rats in the acupuncture group received electroacupuncture on the bilateral KI3 for seven days. The kidneys were perfused with ice-cold saline and all kidney proteins were isolated. After protein sample preparation, two-dimensional gel electrophoresis (2-DE) was performed. The interesting spots were analyzed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). There were nine protein spots with three-fold up-regulation in the kidney after the acupuncture. NAD-dependent isocitrate dehydrogenase and quinone reductase, the proteins involved in energy metabolism, the reduction of endogenous quinones, chemoprotection, and electrophilic stress, were identified. The data indicated that acupuncture at the KI3 of the kidney meridian of the foot shaoyin was able to increase NAD-dependent isocitrate dehydrogenase and quinone reductase expression in the kidney, and supported the relationship between the kidney and KI3.

[Effect of acupuncture combined with intraperitoneal injection of paclitaxel on apoptosis in Lewis mice with lung carcinoma].

OBJECTIVE: To observe the effect of acupuncture in combination with Paclitaxel on apoptosis in Lewis lung carcinoma mice so as to investigate their anti-cancer mechanism. METHODS: A total of 48 mice with inoculated Lewis lung carcinoma (LLC) were randomized into model, acupuncture, medication and acupuncture + medication groups, with 12 cases in each group. LLC model was established by subcutaneously incubating LLC cells into the left hindlimb of the C17 BL/6 mice. "Feishu" (BL13) was punctured and stimulated manually by manipulating the acupuncture needle with small amplitude for 1 main twice again, followed by intraperitoneal injection of Paclitaxel (10 mg/kg). The treatment was given once daily, continuously for 10 days. The tumor weight was recorded, the inhibitory rate of LLC calculated and the apoptosis of LLC cells displayed with TUNEL method. RESULTS: Compared with model group, the tumor weight values of acupuncture, medication and acupuncture + medication groups were significantly lower (P < 0.05), and the tumor inhibitory rates and the apoptotic index (Al) were markedly higher (P < 0.05). Comparison among acupuncture, medication and acupuncture + medication groups showed that the tumor weight values of the later 2 groups were obviously lower than that of acupuncture group, and that of acupuncture + medication group was apparently lower than the weight of medication group (P < 0.05); while the inhibitory rate of tumor and Al in acupuncture + medication group were significantly higher than those in acupuncture and medication groups (P < 0.05). The findings showed that acupuncture, medication and acupuncture + medication could significantly suppress the growth of tumor and promote apoptosis of LLC cells, and the effect of the combined treatment group was significantly superior to that of simple acupuncture and medication. CONCLUSION: Combination of acupuncture and Paclitaxel has a synergistic effect in inhibiting the growth of LLC and accelerating apoptosis of tumor cells.