Intake of n-3 polyunsaturated fatty acids and non-alcoholic fatty liver disease: a cross-sectional study in Japanese men and women.

BACKGROUND/OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is a common condition, in which abnormal amounts of triglycerides accumulate in hepatocytes and is closely related to cardiovascular diseases and diabetes. Dietary fats contribute 15% of fat accumulation in the liver and regulate hepatic lipid metabolism. The supplementation of n-3 polyunsaturated fatty acids (n-3 PUFAs) improves NAFLD. The aim of this study is to assess the cross-sectional association between dietary n-3 PUFAs and NAFLD in Japanese men and women. SUBJECTS/METHODS: Participants were middle-aged, apparently healthy, 296 men and 496 women, who did not drink alcohol and who participated in a general health check-up program. Dietary information from the previous month was obtained by the brief-type self-administered diet history questionnaire. NAFLD was diagnosed if abdominal ultrasonography revealed the presence of fatty liver. RESULTS: The prevalence of NAFLD was 45.3% in men and 17.5% in women. In comparison with the first tertile, multivariate adjusted odds ratios (95% confidence intervals) for the presence of NAFLD in the second and third tertiles for men taking eicosapentaenoic acid (EPA) were 0.59 (0.31-1.14) and 0.45 (0.23-0.90), respectively, (P for linear trend=0.024), and the multivariate adjusted odds ratios (95% confidence intervals) for the presence of NAFLD in the second and third tertiles for men taking EPA+docosahexaenoic acid (DHA) were 0.44 (0.23-0.86) and 0.48 (0.24-0.95), respectively, (P for linear trend=0.035). However, there was no significant relation between NAFLD and each of these nutrients in women. CONCLUSIONS: Dietary EPA and EPA+DHA may be independent and preventive nutrients for NAFLD in Japanese men.

A report of two cases of Werner's syndrome and review of the literature.

Two cases of Werner's syndrome are reported. The first case is that of a man with grey hair since his 20s, and alopecia since aged about 50 years. At the age of 53 years, Werner's syndrome was diagnosed, along with a malignant soft tissue tumour of the hand. The patient underwent ray amputation for the tumour. The subsequent histopathological diagnosis was synovial cell sarcoma, and the patient died of lung metastasis at 15 weeks postsurgery. The second case is that of a woman diagnosed with diabetes mellitus when aged 34 years. At 39 years, a bilateral cataract was diagnosed and at 40 years, diabetic gangrene of the left calcaneal region and calcaneal osteomyelitis necessitated left below-knee amputation. The incidence of Werner's syndrome in Japan is extremely high (1000 of the around 1300 cases reported worldwide) compared to other countries. Most patients develop malignant tumour or arteriosclerosis, the most important complications of this syndrome. The average life expectancy for patients with Werner's syndrome is 46 years. The incidence of epithelial cancer and mesenchymal sarcoma is 10 times that of the general population. The onset of symptoms of Werner's syndrome generally precedes any later symptoms of associated conditions, such as malignant tumour. Therefore, early recognition of Werner's syndrome is important to assist identification of malignant tumours at an early stage in this patient group.

Inhibitory effect of electroacupuncture on murine collagen arthritis and its possible mechanisms.

The influence of electroacupuncture (EA), a traditional Chinese medical treatment, on type II collagen-induced arthritis (CIA) was examined in DBA/IJ mice in vivo. Mice were immunized intradermally twice at a 3-week interval with bovine type II collagen (C II). EA stimulation, begun on day 21 simultaneously with the second immunization, was applied at the acupoint equivalent to GV4 three times a week for 3 weeks. The results showed that EA delayed the onset, attenuated the severity of arthritis, and reduced the anti-collagen antibody level. Furthermore, we investigated the impact of EA on the productions of endogenous interleukin-1 beta (IL-1 beta) and prostaglandin E2 (PGE2), and the levels of IL-1 beta mRNA in splenocytes and synovial tissues from C II immunized mice on day 45 and cyclooxygenase-2 (COX-2) mRNA in lipopolysaccharide (LPS)-stimulated macrophages of normal mice by using reverse transcriptase-polymerase chain reaction (RT-PCR). EA stimulation significantly inhibited the concentrations of splenic endogenous IL-1 beta and serum PGE2. The expression of IL-1 beta mRNA in spleen cells was obviously down-regulated and that in synovial tissues was modestly affected by EA. COX-2 mRNA was highly expressed in cultured peritoneal macrophages when stimulated with LPS. Previous treatment with EA also reduced LPS-stimulated induction of COX-2 mRNA. These data suggest that EA has an inhibitory effect on murine CIA, and the partial mechanism of its therapeutic result may be attributed to inhibiting the productions of IL-1 beta and PGE2 by suppressing the IL-beta and COX-2 gene activations.

Suppressive activity of the chloroform extract of Tripterygium wilfordii Hook f on effector T cell activation during Hymenolepis nana infection in mice.

The chloroform extract of Tripterygium wilfordii Hook f (TWH extract) administered into mice daily at doses of 80.0 to 200.0 micrograms/kg (but not 40.0 micrograms/kg) caused suppression of protective immunity to Hymenolepis nana when the extract was injected subcutaneously during the induction phase of protective immunity. Daily administration of 200.0 micrograms/kg TWH extract, during the course of larval development from challenge, also suppressed protective immunity. Inhibition of protective immunity was only observed in mice that received TWH extract for 6 days at a daily dose of 200.0 micrograms/kg and were challenged 24 h after the final injection. TWH extract did not inhibit formation of effector cells that mediate delayed type hypersensitivity (DTH) to H. nana egg antigen when the extract was administered subcutaneously at a dose of 200.0 micrograms/kg/day for 5 days before cell preparation. However, TWH extract did inhibit DTH effector cell activation when cells prepared from infected, PBS-injected mice were transferred into 200.0 micrograms/kg TWH extract-treated recipient mice. These results strongly indicate that TWH extract cannot inhibit the generation of effector cells but will suppress their function in vivo.

Role of endogenous interferon-gamma on the enhancement of splenic NK cell activity by electroacupuncture stimulation in mice.

Successive electro-acupuncture (EA) stimulation applied to bilateral anterior tibial muscles, where Zusanli (ST36) acupoints are located, once a day (30 min) for 3 successive days significantly enhanced splenic natural killer (NK) cell activity in BALB/c mice. The percentage of splenic NK cells, as measured by flow cytometry, was not affected in these mice. Interferon (IFN)-gamma level in splenic aqueous extract, prepared from the ST36 acupoint-stimulated mice, was significantly higher than that of the controls. In vivo treatment with neutralizing monoclonal antibody against mouse IFN-gamma completely abrogated the increase in splenic NK cell activity induced by ST36 acupoint stimulation. The same stimulation also significantly increased the concentration of splenic beta-endorphin, which coincided with the significant increase in splenic IFN-gamma production. Pre-administration of 10 mg/kg naloxone before initiation of EA stimulation every day reduced the enhancements of NK cell activity and IFN-gamma level. These observations strongly suggest that endogenous IFN-gamma mediates the up-regulation of NK cell activity by EA stimulation at the ST36 acupoints. Furthermore, endogenous beta-endorphin secreted by EA stimulation also plays an important role in the up-regulation of NK cell function, which may be realized through regulating IFN-gamma production.

Antipyretic action of peripheral stimulation with electroacupuncture in rats.

The present study was designed to investigate the antipyretic action of peripheral stimulation with electroacupuncture (EA) in SD rats. EA stimulation was applied for 30 min to the peripheral muscle where the equivalent Quchi (LI11) acupoint is located. We first examined the effects of EA stimulation on fever induced by either lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta) or prostaglandin E2 (PGE2). Intraperitoneal injection of LPS at a dose of 100 micrograms/kg caused a high rectal temperature, which was suppressed by EA stimulation. EA stimulation also inhibited the development of fever induced by IL-1 beta injection either intravenously or into the preoptic area (POA). The rats that received administration of PGE2 into POA developed rapid and high fevers, which were attenuated by EA stimulation. In the second part of the experiment, we investigated the levels of cytokines and PGE2 during the development of fever. The concentrations of IL-6 and PGE2 but not IL-1 beta, in brain and serum were increased by intraperitoneal injection of 100 micrograms/kg LPS. EA stimulation reduced PGE2 levels in both brain and serum in LPS-injected rats, but failed to change the levels of IL-6. Intravenous injection of IL-1 beta produced high brain and serum PGE2 levels, which were also significantly suppressed by EA stimulation. The results suggest that EA stimulation possesses an antipyretic effect through the inhibition of the action of PGE2 in rats.

Influence of moxibustion on collagen-induced arthritis in mice.

The influence of moxibustion, a traditional Chinese medical treatment; on type II collagen-induced arthritis (CIA) was examined in DBA/1J mice in vivo. Mice were immunized intradermally twice at 3-week intervals with bovine type II collagen (C II). The main incidence of arthritis started around day 30 and lasted to day 60 after the first immunization. Moxibustion, using three different regimens, was applied at the acupoint equivalent to GV4 every other day. Moxibustion, from day 0 to day 30 after the first immunization, suppressed the onset and development of arthritis, as well as anti-collagen antibody level. Treatment with moxibustion, from the day 31 to day 60, also resulted in a significant inhibition of progression of arthritis and production of anti-C II antibody. Furtherfore we examined the influence of moxibustion on the established arthritis. Moxibustion given from day 61 to day 120, mildly but significantly decreased the anti-C II antibody level in diseased mice, while the bone erosion and joint destruction were not affected. These results indicate that moxibustion could prevent the incidence and attenuates the development of murine CIA.

Suppressive effects of Tripterygium wilfordii Hook f., a traditional Chinese medicine, on collagen arthritis in mice.

The effect of chloroform extract of Tripterygium wilfordii Hook f. (TWH extract), a traditional immunosuppressive Chinese herb, on type II collagen (C II)-induced arthritis (CIA) in DBA/1J mice was studied. In the first set of experiments, we examined the effect of TWH extract on cellular immune responses to C II. As compared with mice treated with saline, TWH extract administered orally at doses of more than 400 microg kg(-1) once a day for 14 days inhibited the ability of inguinal lymph node cells to produce T cell cytokines interleukin-2 and interferon-gamma when the cells were obtained from mice 21 days after immunization and cultured in vitro with C II. Treatment with TWH extract also inhibited production of macrophage cytokines interleukin-1beta and tumor necrosis factor-alpha in response to in vitro stimulation of lymph node cells with C II. In the second part of the experiment, we evaluated the influence of TWH extract on the incidence and development of arthritis in murine CIA. Mice were immunized twice at a 3-week interval with bovine C II, with TWH extract being given orally once a day for 14 days with four different regimens. A 14-day course of TWH extract treatment at a daily dose of 400 microg kg(-1), which began on the day of the first C II immunization, suppressed the development of arthritis, as well as antibody production and delayed-type hypersensitivity to C II. Treatment with TWH extract, which started on the same day as the booster immunization, also resulted in inhibition of development of arthritis and of immune responses to C II. On the other hand, therapeutic administration with TWH extract did not affect the clinical course of the disease and the immune response to C II.

Stimulation of interleukin-8 production by acidic polysaccharides from the root of Panax ginseng.

The root of Panax ginseng C.A. Meyer, is a well-known important Chinese traditional medicine used as a stomachic, tonic, sedative and as an elixir called Ginseng in China and Japan. The precise mechanism of the biological actions of this plant is not fully understood. In order to elucidate the immunomodulating activities of this plant, we examined the direct effects of four of its components, acidic polysaccharides isolated in previous studies, on cytokine (interleukin-8; IL-8) production by a human monocytic cell line, THP-1, and human blood monocytes in vitro, as IL-8 is a potent inflammatory cytokine involved in neutrophil chemotaxis and activation. We found that one component, ginsenan S-IIA, is a potent inducer of IL-8 production by human monocytes and THP-1 cells, and this induction is accompanied by increased IL-8 mRNA expression.

Enhancement of splenic interferon-gamma, interleukin-2, and NK cytotoxicity by S36 acupoint acupuncture in F344 rats.

The effect of Tsusanli acupuncture point (S36 acupoint) stimulation on splenic natural killer (NK) cytotoxicity was examined in Fischer 344 (F344) rats. Electro-acupuncture stimulation (voltage intensity, 1 to 5 V; duration, 1 ms; frequency, 1 Hz) was applied to bilateral S36 acupoints once a day (1 h) for 3 d. NK cytotoxicity was measured by the standard 4-h 51Cr release assay. Successive acupuncture treatment for 3 d significantly enhanced splenic NK cytotoxicity (p < 0.001) on the first day after final treatment as compared to that of the control. However, similar stimulation to abdominal muscle did not influence splenic NK cytotoxicity. We also examined endogenous cytokine activities in aqueous spleen extracts prepared from acupunctured and control rats. The extracts from rats acupunctured at the S36 acupoint contained high levels of interleukin (IL)-2 and interferon (IFN)-gamma as compared to those of abdominal muscle acupunctured and non-acupunctured control rats (p < 0.01). Furthermore, a significant positive correlation (p < 0.01) was observed between the levels of each cytokine tested and splenic NK cytotoxicity. The same positive correlation was also observed between the levels of IL-2 and IFN-gamma (p < 0.01). These observations indicate that electro-acupuncture stimulation of the S36 acupoint enhances splenic NK cytotoxicity and that IL-2 and IFN-gamma may function, at least in part, in the regulation of NK cell activity in this system.

Molecular cloning of heterotrimeric G-protein alpha-subunits in chicken pineal gland.

The avian pinealocytes have an intrinsic circadian clock function that controls rhythmic synthesis of melatonin, and an environmental light signal can reset the phase of the clock. In addition to the photoendocrine function, the melatonin synthesis of the pinealocytes is regulated by neural signals from sympathetic nerves. Thus the avian pinealocytes show diagnostic characters which seem to represent an evolutionary transition from photosensory cells of lower vertebrates to the neuroendocrinal cells of mammals. To understand the evolutionary background of the regulatory mechanism for the melatonin synthesis in this organ, we screened the chicken pineal cDNA library to find alpha-subunits of heterotrimeric G-proteins involved in the photic and neural regulations. In addition to the transducin-like alpha-subunit (Gt alpha) supposed to mediate the photic pathway, we isolated cDNA clones encoding Gi2 alpha, Gi3 alpha, and Go1 alpha and its splicing variant Go2 alpha. The deduced amino acid sequence of each G alpha had a potential site for pertussis toxin-catalyzed ADP-ribosylation. As it is known that adrenergic receptor-mediated inhibition of melatonin synthesis is blocked by pertussis toxin, the G-proteins identified in the present study are likely to contribute to this neuroendocrine function of the chicken pineal cells.

Inhibition of murine chronic graft-versus-host disease by the chloroform extract of Tripterygium wilfordii Hook f.

The effects of chloroform extract of Tripterygium Wilfordii Hook f (TWH extract) on chronic graft-versus-host disease (GVHD) were examined in a murine experimental model. Chronic GVHD was induced by intravenous transfer of parental DBA/2 spleen cells into unirradiated (C57BL/6 x DBA/2)F1 recipient mice. The effects of TWH extract on GVHD were assessed by measuring both the degree of splenomegaly and the total serum IgE levels 3 weeks after the cell transfer. Subcutaneous administration of TWH extract once a day for 3 weeks suppressed chronic GVHD in a dose-dependent manner. Significant suppression of splenomegaly was first noted in mice treated with 7.5 micrograms/kg of the agent. The maximum inhibition was observed when mice were treated with more than 10.0 micrograms/kg (but not 5.0 micrograms/kg) caused complete suppression of serum IgE hyperproduction. The ability of donor T cells purified from recipient spleen cells to produce interleukin 4 in response to stimulation with anti-CD3 monoclonal antibody was significantly abrogated when recipient mice were treated with 10.0 micrograms/kg of the agent. These results strongly suggest that TWH extract will be an addition to the cohort of immunosuppressive therapies used in solid organ and bone marrow transplantation.

Acupuncture stimulation enhances splenic natural killer cell cytotoxicity in rats.

Acupuncture stimulation of the anterior tibial muscle, which is equivalent to Tsu-sanli point (S36) stimulation and which induces acupuncture analgesia, enhanced YAC-1 cell lytic activity of the splenic natural killer cells (NK activity) from pentobarbital anaesthetized Wistar rats. On the other hand, abdominal muscle stimulation, which does not induce acupuncture analgesia, did not affect NK activity. The serum from rats treated with acupuncture stimulation (acupuncture-stimulated rats) also enhanced the NK activity of other intact rats. The reinforcement effect of serum remained after heat inactivation (56 degrees C, 30 min). The serum from acupuncture-stimulated rats also had more potent complement activity than that from control rats. These results indicate the following considerations: 1) the Tsu-sanli point may be one of the specific points modulating immune activity, 2) this immune modulation system may share a common nervous pathway with the acupuncture analgesia-producing system, 3) acupuncture stimulation may modify NK activity through unknown heat stable humoral factors as well as the nervous system, and 4) acupuncture stimulation may activate the complement system.

Suppressive effects of central opioids on delayed type hypersensitivity to trinitrochlorobenzene: comparative study with morphine and electroacupuncture.

We reported previously that electroacupuncture (Acu) applied to the acu-point equivalent to GV4 in the mouse just before the 2,4,6-trinitrochlorobenzene (TNCB) challenge suppressed the delayed type hypersensitivity (DTH) through endogenous opioidergic systems in the brain, and the pituitary was pivotal in this immunosuppression. The purpose of the present study was to compare the suppressive effects of Acu with those of single, acute doses of morphine on TNCB-DTH in intact and hypophysectomized (HPX) mice. Subcutaneous morphine 10 mg/kg in ddY mice, 30 mg/kg in BALB/c mice or intracisternal morphine 40 micrograms/mouse in BALB/c mice given just before TNCB challenge suppressed (40-53%) the maximal extent of ear swelling at 24 hrs after challenge in intact mice. In HPX mice, the suppressive effects of intracisternal morphine 10 and 100 micrograms/mouse were less pronounced than those observed in intact mice and there was no significant difference between intact and HPX groups. In addition, suppressive effects observed with Acu or subcutaneous morphine (30 mg/kg) were effectively antagonized by pretreatment with intracisternal naloxone at a dose of as low as 2 micrograms/mouse. Naloxone alone had no effect of its own. These results suggest that 1) the activation of opioid receptor-mediated pathways in the brain, which occurs when opioids are endogenously released (Acu) or exogenously given (morphine), is important in the suppression of TNCB-induced DTH, a cell-mediated immune response, and 2) the pituitary is less pivotal in the suppressive effects of acute morphine than in those of Acu.

Involvement of central opioidergic and nonopioidergic neuroendocrine systems in the suppressive effect of acupuncture on delayed type hypersensitivity in mice.

The effect of a single treatment of electroacupuncture (Acu) at early or late stages of the efferent phase on 2, 4, 6-trinitrochlorobenzene (TNCB)-induced delayed type hypersensitivity (DTH) was studied in intact and hypophysectomized (HPX) mice. Acu (2.5 Hz, 15 min) applied to the acu-point equivalent to GV4 at 0, 3, 18 or 21 h after TNCB challenge induced significant suppression (45-73%) of the maximal extent of ear swelling at 24 h after TNCB challenge. An immunosuppressive and antiinflammatory drug, prednisolone 10 mg/kg i.p., also suppressed the DTH to the same extent. Pretreatment with intracisternal injection of naloxone hydrochloride (2 micrograms) significantly blocked the Acu-evoked DTH suppression when Acu treatment was done at 0 or 3 h. On the contrary, naloxone did not block the effect of Acu treatment given at 21 h. In order to examine the potential involvement of the pituitary in the suppression of DTH by Acu, the DTH reaction was examined in HPX mice. Acu failed to produce suppressive response in the HPX mice unless given at 0 h. These findings indicate that Acu treatment at acu-point GV4 during the efferent phase of induced DTH can suppress the DTH through central opioidergic or nonopioidergic systems. The pituitary is apparently pivotal in this immunosuppression and it is suggested that the DTH suppression by Acu may be mediated via activation of the neuroendocrine system.

Suppressive effect of acupuncture on delayed type hypersensitivity to trinitrochlorobenzene and involvement of opiate receptors.

We reported previously that electroacupuncture (Acu) at an acu-point equivalent to GV-4 in mice either enhanced or suppressed the delayed type hypersensitivity (DTH) to 2,4,6-trinitrochlorobenzene (TNCB, picryl chloride) depending on the time of treatment. We report here the suppression of the efferent phase of DTH to TNCB by Acu in mice. In 7- to 9-week-old male BALB/c, C57BL/6 and ddY mice, significant suppression of the DTH was observed when Acu had been applied once per day for three consecutive days before TNCB challenge. When Acu had been applied a single, significant suppression also occurred. Application to another point (at a middle area of the femoral muscle) failed to suppress the DTH to TNCB. This Acu-evoked DTH suppression was blocked dose-relatedly by pretreatment of systemic naloxone hydrochloride, indicating that opioid receptor-mediated mechanisms are involved in this immune response.

Modulation of lipopolysaccharide-induced cytotoxic factor and interferon production by moxibustion in mice.

Pretreatment of mice with moxibustion (Mox) modulated lipopolysaccharide (LPS)-induced endogenous cytotoxic factor (CF) and interferon (IFN) production in serum. CF was measured by the L929 cytotoxicity test and IFN by the cytopathic effect microassay on L929 cells with vesicular stomatitis virus. Significant inhibition of CF activity was observed when Mox and LPS were applied simultaneously. Its potentiation was maximal, about 9 times the control level, when treatment intervals between Mox and LPS were 24-72 hours, and declined thereafter. Mox treatment modified LPS-induced IFN production with a similar biphasic pattern but the onset of modification was delayed. This is the first report of modulation of cytokine production by Mox treatment.

Comparative studies on rat primary cultured and isolated hepatocytes in the evaluation of a therapeutic agents for liver disease.

We have investigated the effect of a therapeutic agent for liver disease, Laennec, on the GOT leakage from freshly isolated and primary cultured rat hepatocytes which were treated with CCl4. By treatment with Laennec together with CCl4, the GOT leakage from isolated hepatocytes increased and that from cultured hepatocytes decreased, compared to those incubated only with CCl4. The results suggest that it is better to use primary cultured hepatocytes than to use freshly isolated hepatocytes to evaluate therapeutic agents for liver disease.

Effects of single moxibustion on phagocytic activity in mice.

Effects of moxibustion stimulation on the phagocytic activity of the reticuloendothelial system in ddY and ICR mice has been studied by using the carbon clearance methods. It was found that moxibustion stimulation induced the enhancement of the phagocytic activity with increased phagocytic indexes (K and a indexes) and lysosomal enzyme activities in mice peritoneal exudate cells and peritoneal macrophages. In addition, the increase of carbon uptake in the Kupffer cells of the liver after carbon injection can be seen by light microscopy when compared with that of nontreated mice. Few big holes on the cell surface of the macrophages obtained from the moxibustion mice were observed in scanning electron microscopical studies. These results suggest that the moxibustion treatment caused the enhancement of the host defence mechanisms in mice.