Electro-acupuncture treatment ameliorates intestinal inflammatory injury in cerebral ischemia-reperfusion rats via regulating the balance of Treg / γδ T cells.

Electro-acupuncture (EA) has an anti-inflammatory role in ischemic stroke, but whether the protective effect of EA involves the regulation of the intestine barrier and Treg/ γδ T cells is unclear. Cerebral ischemia-reperfusion (I/R) injury was induced by middle cerebral artery occlusion(MCAO) for 2 h followed by reperfusion for 24 h. The rats have treated with EA at the "Baihui" acupoint(GV20). Triphenyl tetrazolium chloride (TTC) staining and Longa neurologic score were performed to evaluate the outcomes after ischemic stroke. Inflammatory factor expression levels in the serum, ischemic hemisphere brain, and small intestine were detected by ELISA or RT-qPCR. Additionally, the morphology change of the small intestine was evaluated by analyzing villus height and smooth muscle thickness. Meanwhile, the expression of tight-junction proteins, including Zonula Occludens-1 (ZO-1), Occludin, and Claudin-1, were detected to evaluate the impact of EA on mucosal permeability in the small intestine. The percentages of regulatory T cells (Tregs) (CD45+CD4+Foxp3+) and γδ T cells (CD45+CD4-γδ T+) were measured to assess the effect of EA on intestinal T cells. EA decreased the brain infarction volume and intestine barrier injury in ischemic stroke rats. At the same time, it effectively suppressed the post-stroke inflammation in the brain and small intestine. More importantly, EA treatment increased the percentage of Tregs in the small intestine while reducing the rate of γδ T cells, and ultimately increased the ratio of Treg/ γδ T cells. These results demonstrated that EA ameliorated intestinal inflammation damage by regulating the Treg/ γδ T cell polarity shift and improving the intestine barrier integrity in rats with I/R injury. This may be one of the mechanisms underlying the anti-ischemic injury effects of acupuncture on stroke.

Tissue-specific alterations in expression and function of P-glycoprotein in streptozotocin-induced diabetic rats.

AIM: To investigate the changes of expression and function of P-glycoprotein (P-GP) in cerebral cortex, hippocampus, liver, intestinal mucosa and kidney of streptozocin-induced diabetic rats. METHODS: Diabetic rats were prepared via a single dose of streptozocin (65 mg/kg, ip). Abcb1/P-GP mRNA and protein expression levels in tissues were evaluated using quantitative real time polymerase chain reaction (QRT-PCR) analysis and Western blot, respectively. P-GP function was investigated via measuring tissue-to-plasma concentration ratios and body fluid excretion percentages of rhodamine 123. RESULTS: In 5- and 8-week diabetic rats, Abcb1a mRNA levels were significantly decreased in cerebral cortices and intestinal mucosa, but dramatically increased in hippocampus and kidney. In liver, the level was increased in 5-week diabetic rats, and decreased in 8-week diabetic rats. Abcb1b mRNA levels were increased in cerebral cortex, hippocampus and kidney, but reduced in liver and intestinal mucosa in the diabetic rats. Western blot results were in accordance with the alterations of Abcb1a mRNA levels in most tissues examined. P-GP activity was markedly decreased in most tissues of diabetic rats, except kidney tissues. CONCLUSION: Alterations in the expression and function of Abcb1/P-GP under diabetic conditions are tissue specific, Abcb1 specific and diabetic duration-dependent.

STAT3 but Not STAT5 Contributes to the Protective Effect of Electroacupuncture Against Myocardial Ischemia/Reperfusion Injury in Mice.

Electroacupuncture (EA) can help reduce infarct size and injury resulting from myocardial ischemia/reperfusion (I/R); however, the underlying molecular mechanism remains unknown. We previously reported that STAT5 plays a critical role in the cardioprotective effect of remote ischemic preconditioning (RIPC). Here, we assessed the effects of electroacupuncture pretreatment (EAP) on myocardial I/R injury in the presence and/or absence of Stat5 in mice and investigated whether EAP exerts its cardioprotective effects in a STAT5-dependent manner. Adult Stat5 fl/fl and Stat5-cKO mice were exposed to EAP at Neiguan (PC6) for 7 days before the induction of I/R injury by left anterior descending (LAD) coronary artery ligation. The myocardial infarct size (IS), area at risk, and apoptotic rate of cardiomyocytes were detected. RT-qPCR and western blotting were used to measure gene and protein expression, respectively, in homogenized heart tissues. RNA-seq was used to identify candidate genes and pathways. Our results showed that EAP decreased IS and the rate of cardiomyocyte apoptosis. We further found that STAT5 was activated by EAP in Stat5 fl/fl mice but not in Stat5-cKO mice, whereas the opposite was observed for STAT3. Following EAP, the levels of the antiapoptotic proteins Bcl-xL, Bcl-2, and p-AKT were increased in the presence of Stat5, while that of interleukin 10 (IL-10) was increased in both Stat5 fl/fl and Stat5-cKO. The gene expression profile in heart tissues was different between Stat5 fl/fl and the Stat5-cKO mice with EAP. Importantly, the top 30 DEGs under EAP in the Stat5-cKO mice were enriched in the IL-6/STAT3 signaling pathway. Our results revealed for the first time that the protective effect of EAP following myocardial I/R injury was attributable to, but not dependent on, STAT5. Additionally, we found that EAP could activate STAT3 signaling in the absence of the Stat5 gene, and could also activate antiapoptotic, survival, and anti-inflammatory signaling pathways.

Electroacupuncture improves cardiac function and reduces infarct size by modulating cardiac autonomic remodeling in a mouse model of myocardial ischemia.

BACKGROUND: Sympathetic and parasympathetic nerve remodeling play an important role in cardiac function after myocardial ischemia (MI) injury. Increasing evidence indicates that electroacupuncture (EA) can regulate cardiac function by modulating the autonomic nervous system (ANS), but little is known about its effectiveness on neural remodeling post-MI. OBJECTIVES: To investigate the role of EA in ANS remodeling post-MI. METHODS: Adult male C57/BL6 mice were equally divided into the Control (Ctrl), MI and EA groups after generating the MI model by ligating the left anterior descending (LAD) coronary artery. Echocardiography and 2,3,5-triphenyltetrazolium (TTC) staining were employed to evaluate cardiac function and infarct size after EA treatment for five consecutive days. Serum norepinephrine (NE) levels were measured by ELISA to quantify sympathetic activation. Then, ANS remodeling was detected by immunohistochemistry (IHC), RT-qPCR, and Western blotting. RESULTS: Our preliminary findings showed that EA increased ejection fraction and fractional shortening and reduced infarct area after MI injury. Serum NE levels in the EA group were significantly decreased compared with those in the MI group. IHC staining results demonstrated that the density of growth associated protein (GAP)43 and tyrosine hydroxylase (TH) positive nerve fibers in the EA group were decreased with increased choline acetyltransferase (CHAT) and vesicular acetylcholine transporter (VACHT). Meanwhile, the results verified that mRNA and protein expression of GAP43 and TH were significantly inhibited by EA treatment in the MI mice, accompanied by elevated CHAT and VACHT. CONCLUSIONS: EA treatment could improve cardiac function and reduce infarct size by modulating sympathetic and parasympathetic nerve remodeling post-MI, thus helping the cardiac ANS reach a new balance to try to protect the heart from further possible injury.

[Research and thinking of acupuncture on promoting white adipose tissue browning].

Acupuncture has become an effective approach in clinic for treating obesity, but its mechanism has not been clarified yet. A large number of researches have been conducted on the obesity mechanism in the aspects of neurophysiological regulation, feeding center regulation and peripheral digestion and absorption regulation at home and abroad. But, regarding the main storage site of excess energy, i．e. the remodeling and functional regulation of white adipose tissue (WAT), is still a new field in research. In the paper, focusing on the new filed of weight loss, in view of the promotion of WAT browning through the re-gulation of UCP1 and PPARγ signal pathway with acupuncture, the potential peripheral mechanism of acupuncture was explored on weight loss.

Epigenetic Landscape Changes Due to Acupuncture Treatment: From Clinical to Basic Research.

Acupuncture has been widely used for treating diseases since the ancient days in China, but the mechanism by which acupuncture exerts such powerful roles is unclear. Epigenetics, including DNA methylation, histone modification, and post-transcriptional regulation of miRNAs, is the study of heritable changes in gene expression that do not include DNA sequence alterations. Epigenetics has become a new strategy for the basic and clinical research of acupuncture in the last decade. Some investigators have been trying to illustrate the mechanism of acupuncture from an epigenetics perspective, which has shed new lights on the mechanisms and applications of acupuncture. Moreover, the introduction of epigenetics into the regulatory mechanism in acupuncture treatment has provided more objective and scientific support for acupuncture theories and brought new opportunities for the improvement of acupuncture studies. In this paper, we reviewed the literatures that has demonstrated that acupuncture could directly or indirectly affect epigenetics, in order to highlight the progress of acupuncture studies correlated to epigenetic regulations. We do have to disclose that the current evidence in this review is not enough to cover all the complex interactions between multiple epigenetic modifications and their regulations. However, the up-to-date results can help us to better understand acupuncture's clinical applications and laboratory research.

[Effects of electroacupuncture on food intake and expression of lipid receptors of taste buds in the tongue and hippocampus in obese rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on food intake, body weight, number of taste bud cells and the expression of lipid taste bud receptor (CD36), Gα-gustducin, post-synaptic density protein 95 (PSD95) and neurofilament light chain (NFL) proteins in the tongue or hippocampus in obese rats, so as to explore its mechanism underlying reducing body weight. METHODS: A total of 30 male SD rats were randomly divided into control, model and EA groups (n=10 in each group, 5 rats for H.E. staining and immunohistochemistry, and 5 for Western blot). The obesity model was established by feeding the rats with high fat diet for 11 weeks. Following successful modeling, EA (2 Hz/15 Hz, 1.0-1.2 mA) was applied to "Tianshu" (ST25) for 30 min, once a day, 5 times/week for 5 weeks. The body length, body weight and maximum daily food consumption were recorded every day, followed by calculating the lee's index. Histopathological changes of the circumvallate papillae (CVP) and number of taste bud cells and CD36 were detected by HE staining and immunohistochemistry (IHC), separately. The expression levels of CD36, PSD95 and NFL proteins in the hippocampus were detected by Western blot. RESULTS: The body weight, Lee's index and daily food consumption were significantly higher in the model group than in the control group (P<0.01), and were significantly lowered after EA intervention in comparison with the model group (P<0.01), suggesting an improvement of obesity. H.E. staining displayed that the CVP area and the number of taste bud cells were obviously decreased in the model group in contrast to the control group (P<0.01), and were notably increased in the EA group in contrast to the model group (P<0.05, P<0.01). IHC and Western blot showed that the expression levels of CD36 in the tongue and hippocampus were obviously up-regulated in the model group relevant to the control group (P<0.01, P<0.05), and considerably down-regulated in the EA group relevant to the model group (P<0.05, P<0.01). The expression levels of Gα-gustducin in the tongue, and PSD95 and NFL in the hippocampus were remarkably decreased in the model group relevant to the control group (P<0.01, P<0.05), and significantly increased in the EA group relevant to the model group (P<0.01, P<0.05). CONCLUSION: EA can reduce daily food consumption and body weight in obese rats, which is associated with its effects in down-regulating the expression of CD36 in taste buds and hippocampus, and up-regulating the expression of Gα-gustducin in the tongue, and PSD95 and NFL proteins in the hippocampus. It suggests that EA may regulate the feeding behavior of obese rats by influencing the cognitive memory mechanism involved in CD36 in hippocampus.

Stat5-dependent cardioprotection in late remote ischaemia preconditioning.

Aims: To study the protective effects of late remote ischaemic preconditioning (RIPC) against myocardial ischaemia/reperfusion (I/R) injury and determine whether Stat5 is involved in this protection by using cardiomyocyte-specific Stat5 knockout mice (Stat5-cKO). Methods and results: Mice were exposed to lower limb RIPC or sham ischaemia. After 24 h, the left anterior descending artery (LAD) was ligated for 30 min, then reperfused for 180 min. The myocardial infarct size (IS), apoptotic rate of cardiomyocytes, and serum myocardial enzymes were measured to evaluate for cardioprotective effects. Heart tissues were harvested to determine the cardiomyocytes' anti-apoptotic and survival signaling. When compared with the Stat5fl/fl mice without RIPC, Stat5fl/fl mice with RIPC (Stat5fl/fl+RIPC + I/R) displayed a decreased myocardial IS/LV (16 ± 1.5 vs. 30.1 ± 3.1%, P < 0.01; IS/ area at risk (AAR), 42.2 ± 3.5 vs. 69.2 ± 4.9%, P < 0.01), a reduced cardiomyocyte apoptotic rate (2.1 ± 0.37 vs. 5.5 ± 0.53%, P < 0.01), and lower creatine kinase (CK), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) levels. To the contrary, the Stat5-cKO mice (Stat5fl/fl; Tnnt2Cremice with Doxycycline treatment for 7 days) did not exhibit any effect of RIPC-induced cardioprotection. Activation of STAT5 protein was significantly higher in the Stat5fl/fl+RIPC + I/R group than in the Stat5fl/fl+I/R group, while there was no significant difference between the Stat5-cKO + RIPC + I/R and the Stat5-cKO + I/R group. Further analyses with heart tissues detected decreased protein expressions of cytochrome c (Cyt c) and cleaved Caspase-3 in the Stat5fl/fl+RIPC + I/R mice, along with increased anti-apoptotic molecules, including B-cell lymphoma-extra large (Bcl-xL) and B-cell lymphoma-2 (Bcl-2); such changes were not noted in the Stat5-cKO + RIPC + I/R mice. Additionally, RIPC increased cardiac hypoxia inducible factor-1 (HIF-1α) and interleukin-10 (IL10) protein levels and caused activation of AKT, phosphatidylinositol 3 kinase (PI3K), and vascular endothelial growth factor in the heart of the Stat5fl/fl mice. However, these changes were completely inhibited by the absence of Stat5. Conclusions: These results suggest that RIPC-induced late cardioprotection against myocardial I/R injury is Stat5-dependent and is correlated with the activation of anti-apoptotic signaling and cardiomyocyte-survival signaling.

[Effect of Electroacupuncture on Adenosine Receptor Expression in White Adipose Tissue of Diet-induced Obese Mice].

OBJECTIVE: To investigate the effects of electroacupuncture (EA) on the expression of adenosine receptor (AR) in the white adipose tissue (WAT) of diet-induced obese (DIO) mice, so as to reveal a peripheral mechanism of EA underlying improvement of body weight. METHODS: Forty three-week-old C 57 BL/6 male mice were divided into normal diet group (n=12) and high fat diet group (n=28) randomly, and fed by normal diet and high fat diet for 12 weeks, respectively. In the high fat diet group, mice with body weight over 20% heavier than that of the normal diet group were considered as obese mice. The normal diet mice and the obese mice were divided into normal group (CD, n=5) and normal plus EA group (CD+EA, n=7), or obese group (HFD, n=6) and obese plus EA group (HFD+EA, n=12). The CD+EA group and the HFD+EA group were treated with EA at "Zusanli"(ST 36) and "Neiting"(ST 44, 2 Hz/15 Hz, 0.6-1.0 mA) for 20 min, 6 times a week for 4 weeks. Body weight, ratio of WAT/body weight were calculated, qPCR and Western blot were applied to detect mRNA and protein levels of adenosine receptors in the epididymal adipose tissue (Epi-WAT), respectively. RESULTS: Compared with the normal diet group, high fat diet significantly increased body weight in C 57 BL/6 mice after feeding for 12 weeks (P<0.01); 18 out of 28 mice in the high fat diet group were classified as obesity. After treatment, the body weight and the ratio of Epi-WAT/body weight of the HFD group were increased than those in the CD group (P<0.05), the change of body weight in the HFD group was bigger than that in the CD group (P<0.01). Compared with the HFD group, the body weight and the ratio of Epi-WAT/body weight of the HFD+EA group were decreased after EA (P<0.05), the change of body weight was also significantly increased (P<0.01). No significant differences were found among the four groups in the expression level of A1R mRNA (P>0.05). The expression of A3R mRNA in the HFD group was lower than that in the CD group (P<0.01), while the expressions of A2A R and A2BR proteins were decreased in the HFD group than in the CD group (P<0.01). In comparison with the HFD group, the expression levels of A2AR and A2BR mRNAs and proteins were significantly up-regulated in the HFD+EA group, respectively (P<0.05, P<0.01). CONCLUSIONS: EA intervention is able to reduce the body weight of DIO mice, which Feb be associated with its effects in regulating the expression of A2AR and A2BR in WAT, suggesting a new mechanism of EA in accelerating peripheral WAT metabolism.

Cardioprotective Effect of Electroacupuncture Pretreatment on Myocardial Ischemia/Reperfusion Injury via Antiapoptotic Signaling.

Objectives. Our previous study has used RNA-seq technology to show that apoptotic molecules were involved in the myocardial protection of electroacupuncture pretreatment (EAP) on the ischemia/reperfusion (I/R) animal model. Therefore, this study was designed to investigate how EAP protects myocardium against myocardial I/R injury through antiapoptotic mechanism. Methods. By using rats with myocardial I/R, we ligated the left anterior descending artery (LAD) for 30 minutes followed by 4 hr of reperfusion after EAP at the Neiguan (PC6) acupoint for 12 days; we employed arrhythmia scores, serum myocardial enzymes, and cardiac troponin T (cTnT) to evaluate the cardioprotective effect. Heart tissues were harvested for western blot analyses for the expressions of pro- and antiapoptotic signaling molecules. Results. Our preliminary findings showed that EAP increased the survival of the animals along with declined arrhythmia scores and decreased CK, LDH, CK-Mb, and cTnT levels. Further analyses with the heart tissues detected reduced myocardial fiber damage, decreased number of apoptotic cells and the protein expressions of Cyt c and cleaved caspase 3, and the elevated level of Endo G and AIF after EAP intervention. At the same time, the protein expressions of antiapoptotic molecules, including Xiap, BclxL, and Bcl2, were obviously increased. Conclusions. The present study suggested that EAP protected the myocardium from I/R injury at least partially through the activation of endogenous antiapoptotic signaling.

[Acupuncture Intervention Reduced Weight Gain Induced by Hypoglycemic Agents through Food Intake-related Targets in Central Nervous System].

Clinical practice shows that thiazolidinediones (TZDs) induce weight gain in patients with type-II diabetes mellitus during treatment, which restrains its application and generalization clinically. It has been demonstrated that acupuncture therapy is useful in easing obesity in clinical trials. In the present paper, we summarize the underlying mechanism of weight gain induced by TZDs through food intake-related targets in the central nervous system and analyze the possible effects of acupuncture therapy. Acupuncture therapy is expected to reduce weight gain side effect of TZDs through 1) lowering permeability of blood brain barrier to reduce TZDs concentration in the brain, 2) upregulating the expression of hypothalamic leptin and inhibiting hypothalamic neuropiptide Y expression, and 3) down-regulating activities of peroxisome proliferator-activated receptor to reduce energy intake and fat syntheses.

Effects of Ziziphus jujuba fruit extracts on cytochrome P450 (CYP1A2) activity in rats.

Drug-drug interactions have become a serious problem in the clinic, since plant-based medicines are extensively used. The present study investigated the effects of Ziziphus jujuba fruit (ZJ) extract on the pharmacokinetics of phenacetin, a typical substrate of a cytochrome P450 enzyme CYP 1A2, in rats. The rats were pretreated with the water extract (1.0 g · kg(-1)) or the ethanolic extract (3.6 g · kg(-1)) of ZJ for 10 days, and the pharmacokinetics of phenacetin was investigated after intravenous administration. In an in vitro assay, acetaminophen formation in the hepatic microsomes of ZJ-treated rats was investigated to assess CYP1A2 activity. Our results demonstrated that the treatment with the water and ethanolic extracts of ZJ decreased the plasma concentration of phenacetin and increased the plasma concentration of acetaminophen, resulting in a 43.2% and 15.5% reduction in the AUC0-120 of phenacetin, respectively, and a 53.2% and 64.9% increase in the AUC0-120 of acetaminophen, respectively after intravenous administration. The water or ethanolic extract of ZJ significantly increased the clearance of phenacetin and acetaminophen formation in hepatic microsomes. In conclusion, ZJ extracts displayed effects on the pharmacokinetics of phenacetin and increased the CYP1A2 activity in rats. Therefore, precaution on drug-drug interactions should be taken when ZJ is co-administered with drugs metabolized by CYP1A2, which may result in decreased concentrations of these drugs.

Insulin suppresses the expression and function of breast cancer resistance protein in primary cultures of rat brain microvessel endothelial cells.

The aim of this study was to investigate the role of insulin in the regulation of breast cancer resistance protein (BCRP) function and expression using primary cultured rat brain microvessel endothelial cells (rBMECs) as an in vitro model of the blood brain barrier (BBB). The prazosin uptake assay and western blot analysis were used to assess the function and expression of BCRP, respectively. It was noted that the uptake of prazosin by rBMECs was time-, concentration- and temperature-dependent. The BCRP inhibitors novobiocin and imatinib mesylate significantly increased the uptake of prazosin by the cells in a concentration-dependent manner. The cells were also incubated with sera from diabetic rats for 72 h, serving as a diabetic in vitro model. We found that the uptake of prazosin by rBMECs incubated in the diabetic rat sera was 39.8% of that in normal rat sera, and insulin treatment reversed this decrease. Further results showed that insulin down-regulated the function and expression of BCRP in rBMECs in a concentration-dependent manner. Treatment with an antibody against the insulin receptor abolished the down-regulation of BCRP function and expression that was induced by insulin. These results indicate that insulin suppressed the function and expression of BCRPs in rBMEC primary cultures.

Effect of Huang-Lian-Jie-Du-Decoction on pharmacokinetics of verapamil in rats.

OBJECTIVES: The aim was to investigate the effect of Huang-Lian-Jie-Du-Decoction (HLJDD) on the pharmacokinetic behaviour of verapamil in rats. METHODS: Rats orally received 3.33 g/kg of HLJDD extract for 14 days, and pharmacokinetics of verapamil was investigated after oral and intravenous verapamil. Norverapamil formation for assessing cytochrome P450 3A activity in hepatic and intestinal microsomes of the HLJDD-treated rats was investigated. The inhibitory effect of berberine on the formation of norverapamil in intestinal and hepatic microsomes was also evaluated. KEY FINDINGS: HLJDD treatment increased the plasma concentration of verapamil and decreased the plasma concentration of norverapamil, resulting in a 24% increase in the AUC(0-480) of verapamil and a 25% reduction in the AUC(0-480) of norverapamil after oral administration. However, HLJDD did not alter the pharmacokinetic behaviour of verapamil after intravenous administration. Norverapamil formation showed biphasic kinetics in both intestinal and hepatic microsomes. HLJDD treatment significantly decreased the intrinsic clearance of verapamil in intestinal microsomes, but had no effect on the hepatic metabolism of verapamil. Berberine also inhibited norverapamil formation in both intestinal and hepatic microsomes; the extent of inhibition was larger in intestinal microsomes. CONCLUSIONS: HLJDD displayed a route-dependent effect on the pharmacokinetics of verapamil in rats. HLJDD treatment increased the bioavailability of verapamil partly via inhibiting first-pass verapamil metabolism in the intestine.