To accomplish preliminary Qigong simulation state by acupuncture.

OBJECTIVE: The purpose of the study was to find out how to enter the preliminary Qigong simulation state in a short period of time. METHODS: This is a non-randomized, human experiment with healthy participants. A multi-channel digital physiological data recorder was used to detect whether the participants had entered the Qigong state. Participants were assisted to enter the Qigong state (relaxation, tranquility and naturalness) by being given the sore (sour) feeling produced by acupuncturing Hegu (LI4), and suggestions (repeating words "relax" and "heat" from the hypogastrium). RESULTS: About 72.2% of the participants who had no Qigong experience were found entering the preliminary Qigong simulation state. Most of the physiological parameters measured after the participants entering the Qigong state showed significant changes compared with the baseline data. CONCLUSION: This study revealed that acupuncture-made sore feeling is able to induce the participants to quickly enter the preliminary Qigong simulation state; hence this can be seen as no longer a limited phenomenon, but can be commonly applied to everybody.

Methylmercury induces alveolar macrophages apoptosis.

Through the use of a scanning electronic microscope, it was found that alveolar macrophages treated with 10 micro M of methylmercury for 24 h showed a decrease of surface microvilli, and those treated with 15 micro M of methylmercury underwent deformity and subsequent cell death. To investigate their death patterns, DNA was aspirated from alveolar macrophages and analyzed by electrophoresis. It was discovered that the DNA ladder phenomenon became more obvious as the methylmercury increased in concentration. When 5 mM EGTA was used to eliminate calcium ions, a decrease of the ladder phenomenon was observed. Zinc at 1 mM had a similar inhibitory effect. Moreover, an apoptosis peak was observed on flow cytometry analysis of DNA stained with propidium iodide. Alveolar macrophages stained with Hoechst 33342 demonstrated apoptotic bodies induced by methylmercury. The above data indicate that methylmercury can induce a typical apoptosis in alveolar macrophages. Continuing onto the study of the mechanism of apoptosis as induced by methylmercury in alveolar macrophages, it was discovered that methylmercury could increase the intracellular calcium ion concentration and decrease the pH in alveolar macrophages. To find out which endonuclease was responsible for the methylmercury-induced DNA fragmentation of alveolar macrophages, the nuclear proteins of alveolar macrophages was aspirated and tested under different pH values and in conditions with or without calcium ions, and it was discovered that the endonuclease was calcium dependent without relations to pH values.

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages.

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca(2+)-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-kappaB (NF-kappaB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca(2+)-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca(2+)-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC-alpha and -beta activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-kappaB-specific DNA-protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-kappaB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-kappaB activation. These data suggest that persistent inhibition of ER Ca(2+)-ATPase by TG would influence calcium release from ER Ca2+ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-kappaB activation that induces iNOS expression and NO production.

Casuarinin protects cultured MDCK cells from hydrogen peroxide-induced oxidative stress and DNA oxidative damage.

Casuarinin has been shown to be an antioxidant in acellular experiments. This study was designed to assess the ability of casuarinin, extracted from Terminalia arjuna, to protect cultured Madin-Darby canine kidney (MDCK) cells against H2O2-mediated oxidative stress. A comparison with trolox, a hydrosoluble vitamin E analogue was performed. MDCK cells were pretreated with casuarinin or trolox for 1 h, then exposed to H2O2. After incubation with 0.8 mM H2O2 for 1 h, casuarinin caused a decrease in intracellular peroxide production as shown by dichlorofluorescein (DCF) fluorescence in a concentration-dependent manner. After 3 h exposure to 8 mM H2O2, the percentage of intracellular glutathione (GSH)-negative cells was reduced in the casuarinin-treated group. Addition of 32mM H2O2 to MDCK cells for 3 h induced an increase in the percentage of cells containing 8-oxoguanine but the level of such cells declined in casuarinin-treated cells. These results show that casuarinin is more effective against H2O2-induced oxidative damage than trolox. The data suggest that casuarinin attenuates H2O2-induced oxidative stress, decreases DNA oxidative damage and prevents the depletion of intracellular GSH in MDCK cells.

Early acute necrosis and delayed apoptosis induced by methyl mercury in murine peritoneal neutrophils.

There is growing evidence that heavy metals in general, and mercurial compounds in particular, are immunotoxic. The purpose of this study was to explore the mechanism of MeHg in inducing cell death of mouse peritoneal neutrophils. In this paper we demonstrate that MeHg induces apoptosis and necrosis depending on MeHg concentration. In vitro exposure of mouse peritoneal neutrophils to MeHg resulted in a time- and concentration-dependent cell death. MeHg (15 microM) induced neutrophil necrosis in 13 min. The type of cell death was attributed to necrosis based on cells permeable to the fluorescent dye, propidium iodide and DNA appeared as a smear. With fura-2 microfluorimetric technique, we found that the entry of external Ca2+ into the cytosol played a crucial role in inducing cell necrosis by 15 microM MeHg. However, at lower concentrations, MeHg (10 microM)-induced apoptosis is confirmed by the observation of morphological features characterised by apoptotic bodies and fragmented DNA ladder. MeHg (10 microM) caused an immediate fall in pHi as revealed by the pH-sensitive fluorescent probe 2'7'-bis (carboxyethyl)-5(6)-carboxyfluorescein. We have found that MeHg induced cellular acidification prior to DNA fragmentation so as the other two apoptosis-inducing agents (ZnCl(2) and EGTA). Furthermore, acid-activated endonuclease was increased by MeHg in neutrophils, which we considered to play a possible role in chromatin digestion leading to apoptosis. Taken together, these findings indicate that MeHg induces necrosis at higher concentrations by a rapid increase of [Ca2+]i and apoptosis at lower concentrations by acid activation of endonuclease.

Decrease in Ca2+-activated K+ conductance in differentiated C6-glioma cells.

Ca2+-activated K+ channels were studied in C6-glioma cells in an attempt to correlate changes in expression with cell proliferation and differentiation. In this study, we treated C6-glioma cells with thapsigargin for 48 h. Cell proliferation was markedly inhibited, and cell morphology changed from round to a spindle differentiated shape. Furthermore, intracellular calcium concentration was initially increased during acute treatment with thapsigargin. The internal [Ca2+]i pool was eventually depleted after a 48-h thapsigargin treatment. We have characterized Ca2+-activated K+ currents in less differentiated C6 cells. After differentiation of C6 cells induced by thapsigargin, Ca2+-activated K+ currents were selectively suppressed. These data lend further support to the notion that the expression of Ca2+-activated K+ channels is intimately associated with the proliferation of C6-glioma cells, and the suppression of Ca2+-activated K+ channels coincides with the inhibition of proliferation and subsequent induction of cell differentiation.

The soreness and numbness effect of acupuncture on skin blood flow.

The purpose of this study was to investigate the effect of the De-Qi sensations of acupuncture (sourness-distension and distension-numbness) stimulation. Fifty-two healthy medical student volunteers were given acupuncture at the Hoku (LI-4) acupoint as they were resting. During a test that lasted 30 minutes, their skin blood flow was measured at the Quchi (LI-11) acupoint and their palm temperature was measured. Our results indicated that acupuncture increased blood flow when the De-Qi sensation occurred. If the needle was twirled a few minutes thereafter and the De-Qi feeling again occurred, the same blood flow increase was seen again. If the needle was not twirled, but the test person felt soreness, numbness and heat sensation within a few minutes after needle insertion, the same blood flow increase was also seen. After acupuncture, Quchi did not show continuous increase of blood flow as did Hoku. Hoku acupuncture also increased palm temperature suggesting that the blood flow increased from cutaneous vessel vasodilation. In conclusion, when the test person felt the sore and numb De-Qi sensation, there was an increase of blood flow at the acupuncture points. Thus, our results suggest that increased flow may be one of the mechanisms accounting for meridian system responses during acupuncture.

The anti-inflammatory carbazole, LCY-2-CHO, inhibits lipopolysaccharide-induced inflammatory mediator expression through inhibition of the p38 mitogen-activated protein kinase signaling pathway in macrophages.

1. The present study was undertaken to investigate the anti-inflammatory effects of a synthetic compound, LCY-2-CHO, on the expression of inducible nitric oxide synthase (iNOS), COX-2, and TNF-alpha in murine RAW264.7 macrophages. 2. Within 1-30 microm, LCY-2-CHO concentration-dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E(2) (PGE(2)), and tumor necrosis factor-alpha (TNF-alpha) formation, with IC(50) values of 2.3, 1, and 0.8 microm, respectively. Accompanying inhibition of LPS-induced iNOS, cyclooxygenase-2 (COX-2), and pro-TNF-alpha proteins was observed. 3. Reverse transcription-polymerase chain reaction (RT-PCR) and promoter analyses indicated that iNOS expression was inhibited at the transcriptional level (IC(50)=2.3 microm), that inhibition of COX-2 expression only partially depended on gene transcription (IC(50)=7.6 microm), and that TNF-alpha transcription was unaffected. 4. Transcriptional assays revealed that activation of AP-1, but not NF-kappaB, was concomitantly blocked by LCY-2-CHO. Our results showed that LCY-2-CHO was capable of interfering with post-transcriptional regulation, altering the stability of COX-2 and TNF-alpha mRNAs. 5. Since the 3'-untranslated region (3' UTR) of both COX-2 and TNF-alpha mRNA contains a p38 mitogen-activated protein kinase (MAPK)-regulated element involved in mRNA stability, we assessed the effect of LCY-2-CHO on p38 MAPK. Our data clearly indicated an inhibition (IC(50)=1.7 microm) of LPS-mediated p38 MAPK activity, but not of extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activity. However, kinase assays ruled out a direct inhibition of p38 MAPK action. The selective p38 MAPK inhibitor, SB203580, inhibited the promoter activities of iNOS and COX-2 rather than that of TNF-alpha. 6. In conclusion, LCY-2-CHO downregulates inflammatory iNOS, COX-2, and TNF-alpha gene expression in macrophages through interfering with p38 MAPK and AP-1 activation.

Methylmercury inhibits nitric oxide production mediated by Ca(2+) overload and protein kinase A activation.

The importance of cytosolic free calcium level intracellular Ca(2+), [Ca(2+)]i, in neutrophil activation prompted us to investigate changes in [Ca(2+)]i of neutrophils caused by methylmercury (MeHg), which has been shown to have immunomodulatory properties. We have shown in this paper that MeHg increased [Ca(2+)]i in the mouse peritoneal neutrophil. The L-type calcium channel blocker verapamil can decrease the elevated [Ca(2+)]i caused by 10 microM MeHg, suggesting that Ca(2+)-influx through L-type Ca(2+) channel mediates the effect of MeHg. Moreover, MeHg potently decreased nitric oxide (NO) production but also the protein and mRNA level of NO synthase induced by lipopolysaccharide. Both verapamil (1 microM) and H-89 (10 microM) can antagonize the inhibitory effect of MeHg (10 microM) on NO production. These findings lead us to conclude that MeHg inhibits NO production mediated at least in part by Ca(2+)-activated adenylate cyclase-cAMP-protein kinase A pathway.