A study on the inhibitory effect of Radix Semiaquilegiae extract on human hepatoma HEPG-2 and SMMC-7721 cells.

The main objective of this paper was to investigate the extraction process of ethanol extract of Radix Semiaquilegiae, as well as its inhibitory activity on human hepatoma HepG-2 and SMMC-7721 cells, and to compare the inhibitory effects of different concentrations of ethanol extracts against these two hepatoma cells. Ethanol reflux extraction and ultrasound-assisted extraction with ethanol at room temperature were used in the extraction process, and MTT assay was mainly used in the activity experiment to perform in-vitro anti HepG-2 and SMMC-7721 cell activity screening of ethanol extract, and to calculate the cell inhibition rates of the extracts. The results showed that among the two types of extracts, ethanol reflux extract had more superior antitumour activity to that of the ultrasonic extract, but all of the extracts obtained had certain anti-cancer activities, and the anti-proliferative activity increased with the increase of concentration.

The effects of opioid receptor antagonists on electroacupuncture-produced anti-allodynia/hyperalgesia in rats with paclitaxel-evoked peripheral neuropathy.

Research supports the effectiveness of acupuncture for conditions such as chronic low back and knee pain. In a five-patient pilot study the modality also improved the symptoms of chemotherapy-induced neuropathic pain. Using an established rat model of paclitaxel-induced peripheral neuropathy, we evaluated the effect of electroacupuncture (EA) on paclitaxel-induced hyperalgesia and allodynia that has not been studied in an animal model. We hypothesize that EA would relieve the paclitaxel-induced mechanical allodynia and hyperalgesia, which was assessed 30 min after EA using von Frey filaments. Beginning on day 13, the response frequency to von Frey filaments (4-15 g) was significantly increased in paclitaxel-injected rats compared to those injected with vehicle. EA at 10 Hz significantly (P<0.05) decreased response frequency at 4-15 g compared to sham EA; EA at 100 Hz only decreased response frequency at 15 g stimulation. Compared to sham EA plus vehicle, EA at 10 Hz plus either a µ, δ, or κ opioid receptor antagonist did not significantly decrease mechanical response frequency, indicating that all three antagonists blocked EA inhibition of allodynia and hyperalgesia. Since we previously demonstrated that µ and δ but not κ opioid receptors affect EA anti-hyperalgesia in an inflammatory pain model, these data show that EA inhibits pain through different opioid receptors under varying conditions. Our data indicate that EA at 10 Hz inhibits mechanical allodynia/hyperalgesia more potently than does EA at 100 Hz. Thus, EA significantly inhibits paclitaxel-induced allodynia/hyperalgesia through spinal opioid receptors, and EA may be a useful complementary treatment for neuropathic pain patients.

Serotonin Receptor 2A/C Is Involved in Electroacupuncture Inhibition of Pain in an Osteoarthritis Rat Model.

Osteoarthritis currently has no cure. Acupuncture can benefit patients with knee osteoarthritis by providing pain relief, improving joint function and serving as an effective complement to standard care. However, the underlying mechanisms of its effects are still not completely understood. The present study, an investigation of the effectiveness and mechanisms of electroacupuncture (EA) in attenuating osteoarthritis pain in a rat model, is focused on the involvement of 5-hydroxytryptamine 2A/C (5-HT2A/C) receptors, which play an important role in pain modulation at the spinal level. Osteoarthritis was induced under isoflurane anesthesia by a single intraarticular injection of monosodium iodoacetate (3 mg/50 µL/rat) into one hind leg of each rat. EA was given at acupoints GB 30 and ST 36 on days 1-4 after the injection. Vehicle or ketanserin, a 5-HT2A/C receptor antagonist, was given intraperitoneally (1 mg kg(-1)) or intrathecally (5 µg or 10 µg/10 µL), 30 min before each EA treatment. Assessment of weight-bearing difference between injected and uninjected hind legs was done on days 0, 1-4 and 7. Fos /serotonin and serotonin/Fluorogold double labeling were performed to determine EA activation of serotonergic neurons in the nucleus raphe magnus (NRM) that project to spinal cord. The results showed that EA significantly decreases weight-bearing difference compared to sham EA. Ketanserin pretreatment blocked the analgesic effect of EA but did not influence weight bearing in sham EA control rats. EA also activated serotonergic NRM neurons that project to the spinal cord. These data show that EA inhibits osteoarthritis-induced pain by enhancing spinal 5-HT2A/2C receptor activity.

Acupuncture alleviates the affective dimension of pain in a rat model of inflammatory hyperalgesia.

Although studies demonstrate that electroacupuncture (EA) alleviates the sensory dimension of pain, they have not addressed EA's effect on the affective dimension. An inflammatory pain rat model, produced by a complete Freund adjuvant (CFA) injection into the hind paw, was combined with a conditioned place avoidance test to determine EA's effects and its underpinning mechanism on the affective dimension of pain. CFA-injected rats showed place aversion, i.e. the affective dimension of pain, by spending less time in a pain-paired compartment after conditioning than before, while saline-injected rats did not. CFA rats given EA treatment at GB30 before a post-conditioning test showed no aversion to the pain-paired compartment, indicating that EA inhibited the affective response. Intra-rostral anterior cingulate cortex (rACC) administration of a κ-, but not µ-opioid receptor antagonist, blocked EA action. These data demonstrate that EA activates opioid receptors in the rACC to inhibit the affective dimension of pain.

Rostral ventromedial medulla µ, but not κ, opioid receptors are involved in electroacupuncture anti-hyperalgesia in an inflammatory pain rat model.

It has been reported that intracerebroventricular injection of a µ receptor antagonist blocked 2 but not 100Hz electroacupuncture (EA)-produced analgesia in an uninjured animal model. Because persistent pain changes neural response to external stimulation, we hypothesized that the mechanisms of EA anti-hyperalgesia may be different in persistent pain than in health. Hyperalgesia, decreased paw withdrawal latency (PWL) to a noxious thermal stimulus, was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into the hind paws of rats. Selective antagonists against µ (CTOP: D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2, 6.25 nmol) and κ (Nor-BIN: nor-binaltorphimine, 10 nmol) opioid receptors were infused into the rostral ventromedial medulla (RVM) 10 min before a 30-min EA treatment at acupoint Huantiao (GB30) 1h 30 min post-CFA. PWL was measured before and 2.5 post-CFA. Both 10 Hz and 100 Hz EA-produced anti-hyperalgesia were blocked by intra-RVM µ, but not κ, receptor antagonists. Double immunofluorescence staining demonstrated that µ receptor-containing neurons were GABAnergic and that GABAa receptor-containing neurons were serotonergic in the RVM. The results demonstrated an involvement of RVM µ, but not κ, receptors in EA-produced anti-hyperalgesia. In summary, EA may induce release of endogenous endomorphins that activate µ opioid receptors in GABAnergic neurons to suppress the release of GABA. This removes the tonic inhibition of GABA on serotonergic neurons in the RVM, and activation of these serotonergic neurons inhibits pain. EA may be used as complementary treatment for inflammatory pain.

Involvement of spinal serotonin receptors in electroacupuncture anti-hyperalgesia in an inflammatory pain rat model.

We previously showed that electroacupuncture (EA) activates medulla-spinal serotonin-containing neurons. The present study investigated the effects of intrathecal 5,7-dihydroxytryptamine creatinine sulfate, a selective neurotoxin for serotonergic terminals, the 5-hydroxytryptamine 1A receptor (5-HT1AR) antagonist NAN-190 hydrobromide and the 5-HT2C receptor (5-HT2CR) antagonist SB-242,084 on EA anti-hyperalgesia. EA was given twice at acupoint GB30 after complete Freund's adjuvant (CFA) injection into hind paw. CFA-induced hyperalgesia was measured by assessing hind paw withdrawal latency (PWL) to a noxious thermal stimulus 30 min post-EA. Serotonin depletion and the 5-HT1AR antagonist blocked EA anti-hyperalgesia; the 5-HT2CR antagonist did not. Immunohistochemical staining showed that spinal 5-HT1AR was expressed and that 5-HT2CR was absent in naive and CFA-injected animals 2.5 h post-CFA. These results show a correlation between EA anti-hyperalgesia and receptor expression. Collectively, the data show that EA activates supraspinal serotonin neurons to release 5-HT, which acts on spinal 5-HT1AR to inhibit hyperalgesia.

[Dense innervation of acupoints and its easier reflex excitatory character in rats].

OBJECTIVE: To study the innervating character of tissues around the acupoints and along the meridian course and to analyze the reflex activity correlation between acupuncture points in a given meridian in the rat. METHODS: Forty Wistar and 15 SD rats were used in this study. Electrical activities of microfilaments of the afferent nerves (deep tibial nerve, peroneal nerve and the lateral cutaneous nerve of the leg) were observed for identifying their receptive field and the type of nerve fibers. Nerve stem-antidromic stimulation induced Evan's blue extravasation method was employed to compare the difference of the nerve ending distribution in the acupoint area and non-acupoint area. The reflex activities evoked by electric stimulation of the acupoint were used to analyze the interrelation between acupoint and meridian. RESULTS: Findings showed that a great majority of the afferent nerve endings supplying the tibialis anterior/rectus femoris muscle and the foot skin distributed in an uneven pattern in the sites being in accord with acupoints or with the orbit of meridians. Antidromic stimulation of C-fibers in the deep tibial nerve evoked extravasation of Evan's blue from the plasma into the interstitial fluid, blue foci appeared at the acupoints of the Stomach Meridian and along the orbit of the Stomach Meridian. The special distribution of the afferent nerve endings in the acupoint was also associated with the special reflex activity originating from the acupoints of the muscle group of a given meridian. CONCLUSION: The acupoint is an excitable muscle/skin-nerve complex with greatest concentration of nerve endings. The meridian consists of acupoints that possess a close interaction in physiological reflex activities.

Electroacupuncture activates corticotrophin-releasing hormone-containing neurons in the paraventricular nucleus of the hypothalammus to alleviate edema in a rat model of inflammation.

BACKGROUND: Studies show that electroacupuncture (EA) has beneficial effects in patients with inflammatory diseases. This study investigated the mechanisms of EA anti-inflammation, using a rat model of complete Freund's adjuvant (CFA)-induced hind paw inflammation and hyperalgesia. DESIGN: Four experiments were conducted on male Sprague-Dawley rats (n = 6-7/per group). Inflammation was induced by injecting CFA into the plantar surface of one hind paw. Experiment 1 examined whether EA increases plasma adrenocorticotropic hormone (ACTH) levels. Experiments 2 and 3 studied the effects of the ACTH and corticotropin-releasing hormone (CRH) receptor antagonists, ACTH(11-24) and astressin, on the EA anti-edema. Experiment 4 determined whether EA activates CRH neurons in the paraventricular nucleus of the hypothalammus. EA treatment, 10 Hz at 3 mA and 0.1 ms pulse width, was given twice for 20 min each, once immediately post and again 2 hr post-CFA. Plasma ACTH levels, paw thickness, and paw withdrawal latency to a noxious thermal stimulus were measured 2 h and 5 h after the CFA. RESULTS: EA significantly increased ACTH levels 5 h (2 folds) after CFA compared to sham EA control, but EA alone in naive rats and CFA alone did not induce significant increases in ACTH. ACTH(11-24) and astressin blocked EA anti-edema but not EA anti-hyperalgesia. EA induced phosphorylation of NR1, an essential subunit of the N-methyl-D-aspartic acid (NMDA) receptor, in CRH-containing neurons of the paraventricular nucleus. CONCLUSION: The data demonstrate that EA activates CRH neurons to significantly increase plasma ACTH levels and suppress edema through CRH and ACTH receptors in a rat model of inflammation.

Electroacupuncture attenuates bone-cancer-induced hyperalgesia and inhibits spinal preprodynorphin expression in a rat model.

Cancer pain impairs the quality of life of cancer patients, but opioid intervention can cause significant side effects that further decrease quality of life. Although electroacupuncture (EA) has been used to treat cancer pain, its mechanisms are largely unknown. To examine its effects and underlying mechanisms on cancer pain, we injected AT-3.1 prostate cancer cells into the tibia to induce bone cancer in the male Copenhagen rat. The resulting pain was treated with 10Hz/2mA/0.4ms pulse EA for 30min daily at the point equivalent to the human acupoint GB30 (Huantiao) between days 14 and 18 after the injection. For sham control, EA needles were inserted into GB30 without stimulation. Thermal hyperalgesia, a decrease in paw withdrawal latency (PWL) to a noxious thermal stimulus, and mechanical hyperalgesia, a decrease in paw withdrawal pressure threshold (PWPT), was measured at baseline and 20min after the EA treatment. Preprodynorphin mRNA and dynorphin were determined by RT-PCR and immunohistochemistry, respectively. Thermal and mechanical hyperalgesia developed ipsilaterally between days 12 and 18 after cancer cell inoculation. EA significantly (P<0.05) attenuated this hyperalgesia, as shown by increased PWL and PWPT, and inhibited up-regulation of preprodynorphin mRNA and dynorphin compared to sham control. Intrathecal injection of antiserum against dynorphin A (1-17) also significantly inhibited the cancer-induced hyperalgesia. These results suggest that EA alleviates bone cancer pain at least in part by suppressing dynorphin expression, and they support the clinical use of EA in the treatment of cancer pain.

Electroacupuncture suppresses hyperalgesia and spinal Fos expression by activating the descending inhibitory system.

Although electroacupuncture (EA) is widely used to treat pain, its mechanisms have not been completely understood. The present study investigated the descending inhibitory system involvement in EA action. Inflammatory pain was induced by injecting complete Freund's adjuvant subcutaneously into one hind paw of rats with dorsolateral funiculus lesions and sham-operated rats. EA treatment, 10 Hz at 3 mA, was given twice for 20 min each, once immediately post- and again 2 h post-Freund's adjuvant at GB 30, at the junction of the lateral 1/3 and medial 2/3 of the distance between the greater trochanter and sacral hiatus. For sham EA control, acupuncture needles were inserted bilaterally into GB 30 without electrical or manual stimulation. Paw withdrawal latency to a noxious thermal stimulus was measured at baseline and 20 min after EA treatment. Compared to sham EA, EA significantly (P<0.05, n=9) increased withdrawal latency of the inflamed hind paws in the sham-operated rats but not in those with dorsolateral funiculus lesions, indicating that lesioning blocked EA-produced anti-hyperalgesia. EA, compared to sham EA, also significantly inhibited Fos expression in laminae I-II of the spinal cord in the sham-operated rats (58.4+/-6.5 vs. 35.2+/-5.4 per section) but not in those with dorsolateral funiculus lesions. Further, EA activated serotonin- and catecholamine-containing neurons in the nucleus raphe magnus and locus coeruleus that project to the spinal cord. The results demonstrate that EA inhibits transmission of noxious messages and hyperalgesia by activating supraspinal neurons that project to the spinal cord.

Electroacupuncture attenuates bone cancer pain and inhibits spinal interleukin-1 beta expression in a rat model.

BACKGROUND: Although pain affects the quality of life of cancer patients, current medical treatments are either ineffective or have side effects. In the present study we investigated the effect of electroacupuncture (EA) on cancer-induced hyperalgesia and expression of interleukin-1beta (IL-1beta), upregulation of which is related to the maintenance of persistent pain, in a rat model of bone cancer pain. METHODS: Cancer was induced by injecting AT-3.1 prostate cancer cells into the tibia of male Copenhagen rats. The resulting pain was treated with 10 Hz/2 mA/0.4 ms pulse EA for 30 min daily at the equivalent of the human acupoint GB30 (Huantiao) between Days 14 and 18 after cancer cell inoculation. For sham control, EA needles were inserted into GB30 without stimulation. Thermal hyperalgesia, a decrease in paw withdrawal latency to a noxious thermal stimulus, was measured at baseline and 20 min after EA treatment. IL-1beta and its mRNA were respectively determined by immunohistochemistry and reverse transcription-polymerase chain reaction analysis. RESULTS: Thermal hyperalgesia developed between Days 12 and 18 after cancer cell inoculation. EA significantly (P < 0.05) attenuated this hyperalgesia, increasing paw withdrawal latency from 7.0 +/- 0.3 s to 9.2 +/- 0.4 s, and inhibited the upregulation of IL-1beta and its mRNA compared to the sham control. Intrathecal injection of IL-1 receptor antagonist (IL-1ra, 0.1 mg/rat) also significantly inhibited cancer-induced thermal hyperalgesia. CONCLUSION: The data suggest that EA alleviates bone cancer pain, at least in part by suppressing IL-1beta expression. The results support the clinical use of EA in the treatment of cancer pain.

Corticosterone mediates electroacupuncture-produced anti-edema in a rat model of inflammation.

BACKGROUND: Electroacupuncture (EA) has been reported to produce anti-edema and anti-hyperalgesia effects on inflammatory disease. However, the mechanisms are not clear. The present study investigated the biochemical mechanisms of EA anti-inflammation in a rat model. METHODS: Three experiments were conducted on male Sprague-Dawley rats (n = 7-8/per group). Inflammation was induced by injecting complete Freund's adjuvant (CFA) subcutaneously into the plantar surface of one hind paw. Experiment 1 measured plasma corticosterone (CORT) levels to see if EA regulates CORT secretion. Experiment 2 studied the effects of the adrenal gland on the therapeutic actions of EA using adrenalectomy (ADX) rats. Experiment 3 determined whether a prototypical glucocorticoid receptor antagonist, RU486, affects EA anti-edema. EA treatment, 10 Hz at 3 mA and 0.1 ms pulse width, was given twice, for 20 min each, once immediately after CFA administration and again 2 h post-CFA. Plasma CORT levels, paw thickness, indicative of the intensity of inflammation, and paw withdrawal latency (PWL) were measured 2 h and 5 h after the CFA injection. RESULTS: EA significantly increased plasma corticosterone levels 2 h (5 folds) and 5 h (10 folds) after CFA administration compared to sham EA control, but EA alone in naive rats and CFA alone did not induce significant increases in corticosterone. Adrenalectomy blocked EA-produced anti-edema, but not EA anti-hyperalgesia. RU486 (15 mul, 15 mug/mul), a prototypical glucocorticoid receptor antagonist, also prevented EA anti-edema. CONCLUSION: The data demonstrate that EA activates the adrenals to increase plasma corticosterone levels and suppress edema and suggest that EA effects differ in healthy subjects and in those with pathologies.

Human acupuncture points mapped in rats are associated with excitable muscle/skin-nerve complexes with enriched nerve endings.

As part of our ongoing investigation into the neurological mechanisms of acupuncture, we have tried to correlate the distribution of afferent nerve endings with acupuncture points (AP) in the rat hind limbs. In vivo extracellular microfilament recordings of Aalpha/Abeta/Adelta fibers were taken from peripheral nerves to search for units with nerve endings or receptive fields (RF) in the skin or the muscles. The location of the RFs for each identified unit was marked on scaled diagrams of the hind limb. Noxious antidromic stimulation-induced Evans blue extravasation was used to map the RFs of C-fibers in the skin or muscles. Results indicate that, for both A- and C-fibers, the distribution of RFs was closely associated with the APs. In the skin, the RFs concentrate either at the sites of APs or along the orbit of meridian channels. Similarly, the majority of sarcous sensory receptors are located at the APs in the muscle. Results from our studies strongly suggest that APs in humans may be excitable muscle/skin-nerve complexes with high density of nerve endings.