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Background/Aims: Hypoxic/ischemic injury to the liver is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs (miRNAs) are involved in hypoxic/ ischemic events, and δ-opioid receptor (DOR) is protective against hypoxic/ischemic injury, we asked if pharmacological activation of DOR can alter hypoxic events by regulating miRNA expression in the liver. As the first step, the present work aimed at testing the effect of DOR activation on hepatic miRNA expression in hypoxia. Methods: Male Sprague Dawley rats were exposed to hypoxia (9.5-10% O2) for 1, 5, or 10 days with or without DOR activation. The target miRNAs were selected according to TaqMan low-density array (TLDA) data and analyzed by quantitative real-time PCR. Results: We found that: 1) 1-day hypoxia caused the upregulation of 9 miRNAs (miR-7a-5p, miR-10a-5p, miR-25-3p, miR-26b-5p, miR-122-5p, miR-128a-3p, miR-135b-5p, miR-145-5p, and miR-181a-5p) and the downregulation of 2 miRNAs (miR-34a-5p and miR-182); 2) 5 and 10-days hypoxia altered the expression of 4 miRNAs (miR-34c-5p, miR-184, miR-107-3p and miR192-5p); 3) DOR activation shifted the expression of 8 miRNAs (miR-122-5p, miR-146a-5p, miR-30e-5p, miR-128a-3p, miR-182, miR-192-5p miR-107-3p and miR-184) in normoxic condition; and 4) DOR activation modified hypoxia-induced changes in 6 miRNAs (miR-142-5p, miR-145-5p, miR-146a-5p, miR-204-5p, miR-34a-5p and miR-192-5p). Conclusion: Hypoxia significantly modifies the miRNA profile in the liver, while DOR activation can modify the hypoxic modification. Therefore, it is potentially possible to alter hypoxic/ischemic pathophysiology in the liver through DOR pharmacotherapy.

Background: Hypoxic/ischemic injury to the heart is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs (miRNAs) are involved in hypoxic/ischemic events, and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering miRNA expression in the heart under hypoxic conditions. The present study aimed to test our hypothesis. Methods: Male Sprague Dawley rats were exposed to hypoxia (9.5-10% O2) for 1, 5, or 10 days with or without DOR activation. The target miRNAs were selected from TaqMan low-density array (TLDA) data and were further analyzed by quantitative real-time PCR. Results: We found that: 1) hypoxia alters the miRNA expression profiles depending on the hypoxic duration; 2) DOR activation shifts miRNA expression profiles in normoxic conditions and upregulates miR-128a-3p, miR-134-5p, miR-135a, miR-193a-3p, miR-196a, miR-324-3p, and miR-338; and 3) DOR activation modifies hypoxia-induced changes in miRNA expression and increases the levels of miR-128a-3p, miR-134-5p, miR-135a, miR-193a-3p, miR-196a, miR-324-3p, miR-141, miR-200b, and miR-324-3p. For example, miR-196c-5p decreased by 50% while miR-135a-5p increased 2.9 fold after 10 days under hypoxic conditions. Moreover, DOR activation further strengthened the hypoxia-induced increase of the levels of miR-7a-5p. When DOR was activated using UFP-512, the level of miR-107-3p significantly increased 1 day after the administration of UFP-512, but gradually decreased back to normal under normoxia. Conclusion: Hypoxia significantly modifies the miRNA profile in the heart, which can be mimicked or modified by DOR activation. Defining the targeted pathways that regulate the diverse cellular and molecular functions of miRNAs may provide new insights into potential therapies for hypoxic/ischemic injury of the heart.

Hypoxic/ischemic disruption of ionic homeostasis is a critical trigger of neuronal injury/death in the brain. There is, however, no promising strategy against such pathophysiologic change to protect the brain from hypoxic/ischemic injury. Here, we present a novel finding that activation of delta-opioid receptors (DOR) reduced anoxic Na+ influx in the mouse cortex, which was completely blocked by DOR antagonism with naltrindole. Furthermore, we co-expressed DOR and Na+ channels in Xenopus oocytes and showed that DOR expression and activation indeed play an inhibitory role in Na+ channel regulation by decreasing the amplitude of sodium currents and increasing activation threshold of Na+ channels. Our results suggest that DOR protects from anoxic disruption of Na+ homeostasis via Na+ channel regulation. These data may potentially have significant impacts on understanding the intrinsic mechanism of neuronal responses to stress and provide clues for better solutions of hypoxic/ischemic encephalopathy, and for the exploration of acupuncture mechanism since acupuncture activates opioid system.

Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder, and its pathogenesis has yet to be completely clarified. A fully convincing animal model has not been established for PCOS. In earlier studies, researchers have shown that the exposure of rats to continuous light can induce PCOS; nevertheless, hyperandrogenism, a key characteristic observed in human PCOS, has not been reported previously. In the present study, we found that (1) body weights decreased in female rats in a continuous light environment with both ovarian and uterine augmentation; (2) the estrous cycle in rats under continuous light environment was disordered, and polycystic ovary-like changes occurred, accompanied with fur loss and lethargy; and (3) serum testosterone levels in rats in a continuous light environment significantly increased. Our data suggest that continuous light can lead to the occurrence of PCOS in female rats without the need for drugs; this is a reasonable PCOS animal model that is more consistent with the natural disease state in humans; and poor sleep habits or negligence of sleep hygiene may be an important lifestyle factor in pathogenesis of PCOS.

Delta-opioid receptors (DOR) are involved in cardioprotection under hypoxia/ ischemia. However, the underlying mechanism is not well understood yet. We have recently shown that DOR activation significantly modify the expression of hypoxia-sensitive microRNAs (miRNAs) in the brain and kidney. In extension, we further investigated if DOR activation can regulate cardiac miRNAs in hypoxia. We compared global changes in miRNA gene expression in the rat heart following exposure to hypoxia for 10 days with those in normoxia using a low density miRNA microarray to delineate hypoxia-induced changes in the miRNA expression. The up-regulated miRNAs from the microarray analysis were selected for further investigating their response to hypoxia and DOR activation with a DOR agonist, UFP-512 after 1, 5, and 10 days. We found that 1) miR-376a increased after 1-day hypoxia and progressively increased with hypoxic duration; 2) miR-7b, miR-107, miR-134, miR-135a, miR-193a-3p, miR-196a and miR-324-3p progressively increased after 1-day hypoxia and reached a peak after 5-days; 3) miR-7a, miR-141, miR-196c, miR-200a, miR-200b, miR-203a and miR-324 increased only after 1-day hypoxia and significantly decreased after 5-10 days of hypoxia; 4) miR-128a did not significantly change after 1-10 days of hypoxia. After DOR activation with UFP-512, miR-128a, miR-135a, miR-193a-3p, miR-196a, miR-200a, miR-324-3p and miR-338 continued to increase after 1-day hypoxia, and then decreased after 5-10 days of hypoxia; 5) miR-7b and miR-134, in the condition of DOR activation, increased after 1-day hypoxia and further increased after 5-day hypoxia, while significantly decreased after 10-day exposure. In contrast, some miRNAs like miR-350 decreased in response to hypoxia, while DOR activation almost completely reversed such hypoxic reduction. These results suggest that cardiac miRNAs differentially respond to hypoxic stress with a variable degree directly related to the duration of hypoxic exposure, and DOR activation can regulate such hypoxic responses. Supported by HD034852 , AT004422 , NSFC31071046, CS20102010, ZD201007 , CY 20119004,CY20120003, STCSM10DZ1975800,NBRP09CB522901 & 12CB518502.

Hypoxic/ischemic injury to kidney is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs are differentially involved in hypoxic/ischemic events and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering the microRNA (miRNA) expression in kidney under hypoxic condition. We selected 31 miRNAs based on microarray data for quantitative PCR analysis. Among them, 14 miRNAs were significantly altered after prolonged hypoxia, DOR activation or a combination of both. We found that 1) DOR activation alters miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters miRNA expression depending on the duration of hypoxia; and 3) DOR activation can modify hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by over 70%, while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100%, which was reversed following DOR activation. These first data suggest that hypoxia comprehensively modifies the miRNA profile within the kidney, which can be mimicked or modified by DOR activation. Ascertaining the targeted pathways that regulate the diverse cellular and molecular functions of miRNA may provide new insights into potential therapies for hypoxic/ischemic injury of the kidney.

The efficacy of electroacupuncture (EA) on epilepsy remains to be verified because of previous controversies that might be due to the complexity of the effects induced by different acupoints and stimulation approaches adopted. Therefore, we investigated the effects of EA on epilepsy to determine the specific acupoints and optimal stimulation parameters in this work. Experimental epilepsy was induced by injecting kainic acid to the lateral cerebral ventricle of adult male SD rats. EA with a low-frequency (10 Hz/1 mA) or high-frequency (100 Hz/1 mA) current was applied to the epileptic model for 30 minutes starting at 0.5 hour after the injection. Four pairs of acupoints were tested, that is, Shuigou (DU26) + Dazhui (DU14), Jinsuo (DU8) + Yaoqi (EXB9), Neiguan (PC6) + Quchi (LI11), and Fenglong (ST40) + Yongquan (KI1). We found that (1) low- or high-frequency EA at different acupoints reduced epileptic seizures (P<0.05versus the control) with an exception of low-frequency EA at Neiguan (PC6) and Quchi (LI11); (2) low-frequency EA induced a better effect at Fenglong (ST40) plus Yongquan (KI1) than that of the other acupoints (P<0.05); (3) there is no significant difference in the effects of high-frequency EA at these acupoints; and (4) the high-frequency EA elicited a greater effect than that of low-frequency EA in all groups (P<0.05), with an exception at Jinsuo (DU8) + Yaoqi (EXB9). The EA-induced attenuation appeared 1–1.5 hours after EA with no appreciable effect in the first hour after EA in either the EEG or the behavioral tests. We conclude that EA attenuation of epileptic seizures is dependent on the stimulation parameters and acupoints and that the delay in appearance of the EA effect could be a reflection of the time required by the EA signal to regulate neural function in the central nervous system.

Cardiovascular dysfunction often causes blood/oxygen insufficiency in the kidney that is very sensitive to changes in oxygen/blood delivery. Indeed, hypoxic/ischemic kidney stress is a frequent problem in clinical settings. There is, however, no promising strategy for prevention and treatment of such injury. Since recent studies suggest that microRNAs are differentially involved in hypoxic/ischemic events and delta-opioid receptor (DOR) activation increases antioxidant capacity and protects against hypoxic/ischemic injury, we asked if DOR activation regulates microRNA expression in the kidney under hypoxic condition. We isolated microRNAs from normoxic and hypoxic rat kidneys and used the paired microRNAs for microarray analysis. Thirty-one microRNAs were selected for quantitative PCR analysis based on the microarray data. Among them, 14 microRNAs were significantly altered in response to prolonged hypoxia for 1, 5 and 10 days, DOR activation with UFP-512 (1 mg/kg/day, ip, at days 0, 4 and 8) or a combination of both. Our novel data show that 1) DOR activation shifts miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters the miRNA expression profiles depending on hypoxic durations; and 3) DOR activation modifies hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by >70% (p100% (p

Prolonged hypoxic/ischemic stress may cause cortical injury and clinically manifest as a neurological disability. Activation of the δ-opioid receptor (DOR) may induce cortical protection against hypoxic/ischemic insults. However, the mechanisms underlying DOR protection are not clearly understood. We have recently found that DOR activation modulates the expression of microRNAs (miRNAs) in the kidney exposed to hypoxia, suggesting that DOR protection may involve a miRNA mechanism. To determine if the miRNAs expressed in the cortex mediated DOR neuroprotection, we examined 19 miRNAs that were previously identified as hypoxia- and DOR-regulated miRNAs in the kidney, in the rat cortex treated with UFP-512, a potent and specific DOR agonist under hypoxic condition. Of the 19 miRNAs tested, 17 were significantly altered by hypoxia and/or DOR activation with the direction and amplitude varying depending on hypoxic duration and times of DOR treatment. Expression of several miRNAs such as miR-29b, -101b, -298, 324-3p, -347 and 466b was significantly depressed after 24 hours of hypoxia. Similar changes were seen in normoxic condition 24 hours after DOR activation with one-time treatment of UFP-512. In contrast, some miRNAs were more tolerant to hypoxic stress and showed significant reduction only with 5-day (e.g., miR-31 and -186) or 10-day (e.g., miR-29a, let-7f and -511) exposures. In addition, these miRNAs had differential responses to DOR activation. Other miRNAs like miRs-363* and -370 responded only to the combined exposure to hypoxia and DOR treatment, with a notable reduction of >70% in the 5-day group. These data suggest that cortical miRNAs are highly yet differentially sensitive to hypoxia. DOR activation can modify, enhance or resolve the changes in miRNAs that target HIF, ion transport, axonal guidance, free radical signaling, apoptosis and many other functions.

This chapter summarizes the clinical practice and mechanistic exploration of acupuncture therapy for hypertension and hypotension. Numerous clinical reports have shown that the therapeutic efficacy of acupuncture on abnormal blood pressure is dependent on the acupoints, acupuncture manipulation or stimulation parameters in the case of electroacupuncture, applying window, times of treatment, and other factors. The mechanistic research has established the idea that acupuncture signals initiated at the acupoints are transferred to the brain through the nervous pathway, thus, modulating multiple neurotransmitter systems at different levels. The output signaling cascades correct abnormal blood pressure, mainly through neural regulation. In addition, acupuncture-induced correction of abnormal blood pressure is also observed to be dependent on the endocritic, humoral, and dielectric regulation. Collectively, acupuncture induces a comprehensive modulation of the cardiovascular activity, consequently, adjusting the blood pressure. Lastly, we will comment on several unsolved issues and future directions of the research concerning acupuncture treatment for abnormal blood pressure.

This chapter reviews the clinical and laboraotry research on acupuncture therapies for arrhythmia and other cardiac diseases (e.g., coronary atherosclerotic heart disease, heart failure, and viral myocarditis). Clinical data suggest that the efficacy of acupuncture therapy varies depending on multiple factors, including the type of diseases, acupoints selected, and the manner of manipulation used. In general, acupuncture induces a much better efficacy in the patients with arrhythmia related to neural dysfunction than in those with pathological changes in the heart. The mechanistic research shows that acupuncture signal is mainly transferred to the central nervous system by afferent nerve fibers. The input signal leads to serial changes in electrical and chemical activities in the brain. Subsequently, the body corrects cardiovascular dysfunctions through neural regulation, endocrine secretion, humoral and dielectric regulation, intracellular modulation of signal transduction, and even gene expression. Thus, acupuncture induces a therapeutic effect on cardiovascular system through an integrated and complex mechanism.

The use of opioid analgesics has a long history in clinical settings, although the functions of opioid receptors, especially their role in the brain, are not well understood yet. Recent studies have generated abundant new data on opioid receptor-mediated functions and the underlying mechanisms. The most exciting finding in the past decade is probably the neuroprotection against hypoxic/ischemic stress mediated by delta-opioid receptors (DOR). An up-regulation of DOR expression and the release of endogenous opioids may increase neuronal tolerance to hypoxic/ischemic stress. The DOR signal triggers, depending on stress duration and severity, different mechanisms at multiple levels to preserve neuronal survival, including the stabilization of ionic homeostasis, an increase in pro-survival signaling (e.g., PKC-ERK-Bcl 2) and the enhanced anti-oxidative capacity. Recent data on DOR-mediated neuroprotection provide us a new concept of neuroprotection against neurological disorders and have a potentially significant impact on the prevention and treatment of some serious neurological conditions, such as stroke.