Inhibitory effects of Keishi-bukuryo-gan on free radical induced lysis of rat red blood cells

Keishi-bukuryo-gan (KBG) prevents the progression of atherosclerosis in cholesterol-fed rabbits by its antioxidative effect. The present study was to test our hypothesis that ingestion of KBG would protect red blood cell (RBC) membranes from free radical induced oxidation if polyphenolic antioxidants in KBG could be absorbed and circulated in the blood. When incubated with a RBC suspension, KBG and four of five herb medicines constituting KBG provided strong protection for RBC membranes against haemolysis induced by 2,2-azo-bis (2-amidinopropane) dihydrochloride (AAPH), an azo free radical initiator. The inhibitory effect was dose dependent at concentrations of 100-1000 microg/mL. Furthermore, the ingestion of 200 mg of KBG was associated with a significant decrease in susceptibility of RBC to haemolysis in rats.     

Effect of angiotensin converting enzyme inhibition on myocardial phosphoinositide metabolism visualised with 1-[1-11C]-butyryl-2-palmitoyl-rac-glycerol in myocardial infarction in the rat

We recently reported that myocardial phosphoinositide (PI) metabolism can be visualised by 1-[1-11C]-butyryl-2-palmitoyl-rac-glycerol (11C-DAG) in rats with myocardial infarction (MI). Angiotensin II, the receptors for which are expressed predominantly in infarcted areas with active fibrogenesis rather than in non-infarcted regions, is involved in the upstream signalling systems of PI metabolism and plays an important role in the process of left ventricular (LV) remodelling after MI. We therefore hypothesised that the distribution of 11C-DAG after MI may be affected by the inhibition of angiotensin converting enzyme, which is one of the most important factors in the development of LV remodelling after MI. Rats were injected with 11C-DAG after 3 or 10 weeks of treatment with captopril or no treatment following coronary artery ligation, and quantitative autoradiography was performed. Cells occupying the infarcted region were identified by immunohistochemistry. Compared with untreated rats, treatment with captopril for 3 weeks after MI elicited a reduction in the 11C-DAG uptake in the infarcted region (P<0.05) but not in the non-infarcted region, and was associated with a 22% decrease in the heart weight/body weight ratio. The thallium-201 distribution in the infarcted area was similarly low in the rats with and rats without the 3-week captopril treatment after MI. Abundant macrophages and myofibroblasts occupied the infarcted area in both rats with and rats without the captopril treatment for 3 weeks after MI. The 11C-DAG radioactivity in the infarcted region in the untreated rats was lower 10 weeks after MI than 3 weeks after MI (P<0.01). This finding was in agreement with the results of immunohistochemistry demonstrating that the number and size of macrophages and myofibroblasts were remarkably reduced in rats 10 weeks after MI compared with 3 weeks after MI. Captopril treatment for 10 weeks after MI did not decrease the 11C-DAG radioactivity in the infarcted area further. These data suggest that 11C-DAG is useful for visually detecting regions with activated PI metabolism after MI, and that captopril reduces PI metabolism in the infarcted region in the relatively early phase of MI, which might contribute to the attenuation of ventricular remodelling.     

Cholinergic inputs to rostral ventrolateral medulla pressor neurons from hypothalamus

The rostral ventrolateral medulla (RVLM) has cholinergic mechanisms responsible for pressor responses. Stimulation of the hypothalamic paraventricular nucleus (PVN) causes an increase of arterial pressure via activation of neurons in the RVLM. In this study, we examined whether PVN stimulation causes a pressor response via activation of cholinergic mechanisms in the RVLM. Male Wistar rats were used and they were anesthetized, paralyzed and artificially ventilated. Electrical stimulation of the PVN produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine and the cholinesterase inhibitor physostigmine into the RVLM inhibited and potentiated, respectively, the pressor response induced by PVN stimulation. PVN stimulation also increased the firing rate of RVLM barosensitive neurons and the increase in the firing rate was inhibited and potentiated by scopolamine and physostigmine, respectively, iontophoretically applied on neurons. Microinjection of L-glutamate into the PVN produced a release of ACh in the RVLM. The inhibitory amino acid gamma-aminobutyric acid injected into the lateral parabrachial nucleus (LPBN) inhibited the pressor response induced by PVN stimulation. These results suggest that PVN stimulation causes an increase in arterial pressure via activation of cholinergic inputs in the RVLM. It appears that the pressor response is mediated, at least in part, via cholinergic inputs from the LPBN.     

A quantitative evaluation of active gelatinolytic sites in uterine endometrioid adenocarcinoma using film in situ zymography: association of stronger gelatinolysis with myometrial invasion.

Collagen matrix degradation by malignant tumor cells plays an essential role in the process of tumor invasion and metastasis. The purpose of this study was to detect in situ gelatinase activity in endometrioid adenocarcinomas of the uterine corpus. In order to carry out quantitative evaluation, autoexposure time (AET) on gelatin-coated film (film in situ zymography: FIZ) was measured. The gelatinase activity was located primarily within cancers and was prominently suppressed by the addition of a chelating agent to the film. This suggests that matrix metalloproteinases (MMPs) play an important role in the gelatinase activity. The gelatinase activity in the normal endometrium is almost negligible, despite positive immunoreactivity for MMP-2 and -9. Tumor tissues that had invaded more than half of the myometrium showed significantly higher activity than those that had invaded less than half. There was no significant difference in gelatinase activity among tumor stages, grades, vessel invasion or immunoreactivity for MMPs, with the exception that stage 2b cancers showed higher activity than stage 1a. The study suggested that the level of MMP-mediated gelatinolysis is an important factor for myometrial invasion in uterine endometrioid adenocarcinoma. Thus, a quantitative assessment of active gelatinolysis using FIZ and AET should be a useful tool in evaluating in situ matriolytic activity in local myometrial invasion by uterine endometrioid adenocarcinoma.     

A Quantitative Evaluation of Active Gelatinolytic Sites in Uterine Endometrioid Adenocarcinoma Using Film in situ Zymography: Association of Stronger Gelatinolysis with Myometrial Invasion

Collagen matrix degradation by malignant tumor cells plays an essential role in the process of tumor invasion and metastasis. The purpose of this study was to detect in situ gelatinase activity in endometrioid adenocarcinomas of the uterine corpus. In order to carry out quantitative evaluation, autoexposure tune (AET) on gelatin-coated film (film in situ zymography: FIZ) was measured. The gelatinase activity was located primarily within cancers and was prominently suppressed by the addition of a chelating agent to the film. This suggests that matrix metalloproteinases (MMPs) play an important role in the gelatinase activity. The gelatinase activity in the normal endometrium is almost negligible, despite positive immunoreactivity for MMP-2 and -9. Tumor tissues that had invaded more than half of the myometrium showed significantly higher activity than those that had invaded less than half. There was no significant difference in gelatinase activity among tumor stages, grades, vessel invasion or immunoreactivity for MMPs, with the exception that stage 2b cancers showed higher activity than stage la. The study suggested that the level of MMP-mediated gelatinolysis is an important factor for myometrial invasion in uterine endometrioid adenocarci- noma. Thus, a quantitative assessment of active gelatinolysis using FIZ and AET should be a useful tool in evaluating in situ matriolytic activity in local myometrial invasion by uterine endometrioid adenocarcinoma.     

Interleukin-6 (IL-6) production in carcinoma of the cervix

Interleukin-6 (IL-6) is a pleiotropic cytokine that is not only a mediator in major immunologic reactions but also a growth factor of keratinocytes. We studied the IL-6 secretion in vitro of 15 human cell lines derived from both squamous cell carcinoma (SCC) and adenocarcinoma of the uterine cervix. Four of the eight well differentiated SCC secreted a large amount (> 1500 pg/48 h/10⁶ cells) of IL-6 in nude mice. In contrast, poorly differentiated SCC cell lines and all of the 7 adenocarcinoma cell lines secreted a small amount (< 500 pg/48 h/10⁶ cells of IL-6). The expression of IL-6 mRNA of the cell lines correlated well with their IL-6 secretion potential. However, the expression of IL-6 receptor did not correlate with the IL-6 secretory potential. We also studied the IL-6 secretion of freshly isolated normal squamous epithelium and of dysplastic epithelium. In culture, two normal squamous epithelia secreted a large amount (> 2000 pg/48 h/10⁶ cells), whereas 8 dysplasia epithelia secreted an extremely small amount (< 10 pg/48 h/10⁶ cells). About one-third of patients with SCC had a raised serum IL-6 value. IL-6 production may help to differentiate between SCC and adenocarcinoma of the uterine cervix. IL-6 regulation seems to change in the course of SCC carcinogenesis.