[Advances in the relationship between lung cancer and microbiota].

Interaction exists in lung cancer and microbiota. Lung microecological homeostasis can improve the immune tolerance, enhance immune suppression, and inhibit inflammatory responses, to reduce the lung cancer; while lung cancer can lead to pulmonary microecological imbalance, change the lung environment, and promote tumor cell proliferation. Therefore, modulating microbial flora and microecological immunotherapy may be a potential and preventive treatment for lung cancer, to restore tumor immunosuppression and improve patient survival. However, the individual differences in the lung microecology, because of different genetics, ethnic characteristics, and dietary habits, increasing the difficulty of precise diagnosis and treatment, which is also the current bottleneck in the application of microecological immunotherapy. Otherwise, the effectiveness of regulatory measures such as probiotics, prebiotics or antimicrobials is questionable. The research on microbial flora is still in its infancy, and further exploration is needed to form a standardized, effective, and precise treatment plan. So, standardized, effective, and precise microbial flora treatment strategies need to be further explored.

[Clinical study of circulating tumor cells in monitoring the efficacy of neoadjuvant immunotherapy for non-small cell lung cancer].

Objective: To investigate the changes of CTCs and the correlation between the changes of CTCs and the clinical efficacy of neoadjuvant immunotherapy for non-small cell lung cancer (NSCLC). Methods: A retrospective case-control study was conducted to collect the data of 23 patients with NSCLC who received neoadjuvant immunotherapy in the Third Xiangya Hospital from June 2018 to December 2019. They were 35-76 years old with a median age of 52 years old, including 13 male patients and 10 female patients. The CTCs value, evaluation results from response evaluation criteria in solid tumor (RECIST) and major pathological response were evaluated before treatment, after neoadjuvant immunotherapy and after operation. Mann Whitney U test was used for the comparison between the two groups, Wilcoxon test was used for the comparison of association samples, and Kruskal Wallis test was used for the comparison between multiple samples. Results: The CTCs value was positively correlated with tumor progression, that the CTCs value of ⅡB group, ⅢA group and ⅢB group was 10.69 (3.87) FU/3 ml, 12.90 (2.24) FU/3 ml and 16.04 (3.43) FU/3 ml, and the difference was statistically significant (χ2=7.829, P=0.020). Then CTCs decreased to 7.60(4.79) FU/3 ml significantly (Z=4.197,P=0.000), and decreased to 6.22(2.80) FU/3 ml significantly again after surgery(Z=-2.950,P=0.005). In RECIST results, the CTCs value of CR group, PR group and SD group was 12.90(3.79)FU/3 ml, 12.52(3.96) FU/3 ml and 13.58(5.11) FU/3 ml,and no significant difference before treatment (χ²=1.806, P=0.405). After neoadjuvant immunotherapy, the CTCs of CR group decreased to 6.22(3.87) FU/3 ml significantly (Z=-4.950, P= 0.000), and also PR group to7.32(4.31) FU/3 ml (Z=-3.180, P=0.001) or SD group to (Z=-2.023, P=0.043). There was no significant difference between CR group and PR group (Z=-0.838, P=0.402), but significant difference between SD group and CR/PR group (Z=-1.922, P=0.050). After operation, the CTCs of CR, PR and SD group decreased to 6.09(3.43) FU/3 ml, 6.40(1.82) FU/3 ml and 9.20(5.16) FU/3 ml,and there was no significant difference to preparation in CR group and PR group, but significant difference in SD group (Z=-2.023, P=0.043). There was no significant difference between CR group and PR group (Z=-1.134, P=0.257), but significant difference between SD group and CR/PR group (Z=-1.624, P=0.014). Before treatment,CTCs of MPR group and non-MPR group were 11.98(4.14) FU/3 ml and 13.54(4.76) FU/3 ml,and there was no significant difference between them (Z=-1.354, P=0.176). After neoadjuvant immunotherapy, the CTCs of MPR group decreased to 6.36(2.65) FU/3 ml significantly (Z=-2.934, P=0.001) and also in non-MPR group to 10.88(2.80) FU/3 ml (Z=-2.840, P=0.003); but there was significant difference between MPR group and non-MPR group (Z=-3.693, P=0.000), and also the change of CTCs between two groups (Z=-2.770, P=0.006). After operation, the CTCs of MPR group decreased to 5.40(1.33) FU/3 ml insignificantly (Z=-0.533, P=0.594) but significantly to 7.05(3.80) FU/3 ml in non-MPR group (Z=-2.734, P=0.030), and significant difference between them (Z=-1.900, P=0.011). Conclusion: The value of CTCs is negatively correlated with the efficacy (RECIST and MPR) of neoadjuvant immunotherapy for NSCLC, which can be used for clinical efficacy evaluation of neoadjuvant immunotherapy.

Risk factors for periodic breathing in acute stroke: tracheobronchial infection.

BACKGROUND AND PURPOSE: Despite evidence from clinical and population studies, the aim of the present study was to suggest that multiple factors contribute to periodic breathing (PB). However, little information has been focused on episodes of tracheobronchial infections (TBI) preceding PB onset. METHODS: Thirty subjects with acute stroke who had PB and 41 subjects with acute stroke that of a sex- and age-matched control group without PB were retrospectively evaluated. Stroke location, extent of stroke (demonstrated on CT or MRI), and characteristics of TBI before PB were assessed. PB diagnosis was carried out using a portable device and a pulse oximeter. Risk factors for patients with PB were compared with those without PB by univariate and multivariate analysis. RESULTS: Twenty-four TBI in 30 patients with PB and 11 TBI in 41 patients with non-PB were diagnosed. There was no significant difference in age, sex, body mass index, stroke type, stroke location, or underlying diseases between the two groups (P > 0.05). There was a significant difference in snoring, first recurrent stroke, Glasgow Coma Scale, congestive heart failure, TBI, and inflammatory responses between the PB and non-PB group (P < 0.05). Multiple logistic regression analyses showed a difference in the prevalence of snoring (OR = 10.813, CI = 2.131-54.866, P < 0.01), TBI (OR = 5.313, CI = 1.241-22.740, P < 0.05), and inflammatory responses (OR = 7.315, CI = 1.253-43.123, P < 0.05) between the two groups. CONCLUSIONS: In addition to snoring, TBI and inflammatory responses are the two independent predictors for PB in patients with acute stroke. Clinicians should be encouraged to systematically evaluate TBI and inflammatory responses before PB in patients with acute stroke.

Lactobacillus paracasei PS23 reduced early-life stress abnormalities in maternal separation mouse model.

Maternal separation (MS) has been developed as a model for inducing stress and depression in studies using rodents. The concept of the gut-brain axis suggests that gut health is essential for brain health. Here, we present the effects of administration of a probiotic, Lactobacillus paracasei PS23 (PS23), to MS mice against psychological traits including anxiety and depression. The administration of live and heat-killed PS23 cells showed positive behavioural effects on MS animals, where exploratory tendencies and mobility were increased in behavioural tests, indicating reduced anxiety and depression compared to the negative control mice (P<0.05). Mice administered with both live and heat-killed PS23 cells also showed lower serum corticosterone levels accompanied by higher serum anti-inflammatory interleukin 10 (IL-10) levels, compared to MS separated mice (P<0.05), indicating a stress-elicited response affiliated with increased immunomodulatory properties. Assessment of neurotransmitters in the brain hippocampal region revealed that PS23 affected the concentrations of dopaminergic metabolites differently than the control, suggesting that PS23 may have improved MS-induced stress levels via neurotransmitter pathways, such as dopamine or other mechanisms not addressed in the current study. Our study illustrates the potential of a probiotic in reversing abnormalities induced by early life stress and could be an alternative for brain health along the gut-brain axis.

Anti-inflammatory effects of dimemorfan on inflammatory cells and LPS-induced endotoxin shock in mice.

BACKGROUND AND PURPOSE: Dimemorfan (a sigma1 receptor agonist) showed neuroprotective properties in animal models of inflammation-mediated neurodegenerative conditions, but its effects on inflammatory cells and systemic inflammation remain unclear. EXPERIMENTAL APPROACH: The effects of dimemorfan on phorbol-12-myristate-13-acetate (PMA)- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)- induced neutrophils and lipopolysaccharide (LPS)-activated microglial cells, as well as LPS-induced endotoxin shock in mice were elucidated. KEY RESULTS: Dimemorfan decreased PMA- and fMLP-induced production of reactive oxygen species (ROS) and CD11b expression in neutrophils, through mechanisms independent of sigma1 receptors, possibly by blocking ROS production and G-protein-mediated intracellular calcium increase. Dimemorfan also inhibited LPS-induced ROS and nitric oxide (NO) production, as well as that of monocyte chemoattractant protein-1 and tumour necrosis factor-alpha (TNF-alpha), by inhibition of NADPH oxidase (NOX) activity and suppression of iNOS up-regulation through interfering with nuclear factor kappa-B (NF-kappaB) signalling in microglial cells. Treatment in vivo with dimemorfan (1 and 5 mg kg(-1), i.p., at three successive times after LPS) decreased plasma TNF-alpha, and neutrophil infiltration and oxidative stress in the lung and liver. CONCLUSIONS AND IMPLICATIONS: Our results suggest that dimemorfan acts via sigma1 receptor-independent mechanisms to modulate intracellular calcium increase, NOX activity, and NF-kappaB signalling, resulting in inhibition of iNOS expression and NO production, and production of pro-inflammatory cytokines. These effects may contribute its anti-inflammatory action and protective effects against endotoxin shock in mice.

Cryptotanshinone inhibits chemotactic migration in macrophages through negative regulation of the PI3K signaling pathway.

BACKGROUND AND PURPOSE: Cryptotanshinone, the major tanshinone isolated from Salvia miltiorrhiza Bunge, exhibits anti-inflammatory activity. However, there is no report on the effect of cryptotanshinone on recruitment of leukocytes to inflammatory sites. We therefore assessed the effects of cryptotanshinone on macrophage chemotaxis. EXPERIMENTAL APPROACH: Macrophage migration induced by complement 5a (C5a) or macrophage inflammatory protein-1alpha (MIP-1alpha) was measured in vitro. Intracellular kinase translocation and phosphorylation was assessed by Western blotting. KEY RESULTS: RAW264.7 cell migration towards C5a (1 microg ml(-1)) was significantly inhibited by cryptotanshinone (1, 3, 10 and 30 microM) in a concentration-dependent manner. Primary human macrophages stimulated by C5a were similarly inhibited. C5a-evoked migration in RAW264.7 cells was significantly suppressed by wortmannin (phosphatidylinositol 3-kinase (PI3K) inhibitor), PD98059 (MEK1/2 inhibitor) and SB203580 (p38 mitogen-activated protein kinase (MAPK) inhibitor), but not by SP600125 (c-Jun N-terminal kinase (JNK) inhibitor), suggesting that activation of PI3K, ERK1/2 and p38 MAPK signal pathways was involved in responses to C5a. Western blotting revealed that cryptotanshinone significantly inhibited PI3K-p110gamma membrane translocation and phosphorylation of Akt (PI3K downstream effector protein) and ERK1/2 induced by C5a. However, neither p38 MAPK nor JNK phosphorylation was affected by cryptotanshinone. Wortmannin significantly attenuated C5a-induced PI3K-p110gamma translocation, Akt and ERK1/2 phosphorylation. PD98059 suppressed ERK1/2 phosphorylation but failed to modify PI3K-p110gamma translocation by C5a stimulation. Furthermore, MIP-1alpha-induced cell migration and PI3K-p110gamma translocation were also inhibited by cryptotanshinone in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: Inhibition of macrophage migration by cryptotanshinone involved inhibition of PI3K activation with consequent reduction of phosphorylation of Akt and ERK1/2.

Study on the vascular reactivity and alpha 1-adrenoceptors of portal hypertensive rats.

1. Vascular hyporesponsiveness in portal hypertension has been demonstrated to various vasoconstrictors including noradrenaline (NA). The present study aimed to determine whether the attenuated vascular responsiveness to NA is due to a change in the affinity or the number of alpha 1-adrenoceptors. 2. Partial portal vein ligation (PVL) was performed in Sprague-Dawley rats to produce portal hypertension. Vascular responsiveness to NA was assayed in portal vein, mesenteric artery or tail artery. The affinity and number of alpha 1-adrenoceptors were determined by specific binding of [125I]-HEAT (2-beta-4-hydroxy-3-iodophenyethyl-aminomethyltetralone). 3. In the presence of yohimbine (10(-7) M, an alpha 2-adrenoceptor antagonist), propranolol (10(-6) M, a beta-adrenoceptor antagonist), and two catecholamine uptake inhibitors, desipramine (10(-7) M) and normetanephrine (10(-6) M), the maximum responses to NA were decreased in all three blood vessels of PVL rats: 45% decrease in portal vein, 25% in mesenteric artery and 18% in tail artery. 4. The EC50 values of NA and the pA2 values of prazosin, an alpha 1-adrenoceptor antagonist, in all three blood vessels were not significantly different between sham-operated and PVL rats. 5. The KD and Bmax values for specific binding of [125I]-HEAT or the Ki values for NA in the crude membrane preparations of either mesenteric artery or tail artery were also not significantly different between the two groups. 6. It is concluded that the vascular hyporesponsiveness to NA in the mesenteric artery or tail artery of PVL rats is not due to changes in the affinity or number of alpha 1-adrenoceptors.

Protective effects of baicalein and wogonin against benzo[a]pyrene- and aflatoxin B(1)-induced genotoxicities.

To evaluate the protective effects of baicalein and wogonin against benzo[a]pyrene- and aflatoxin (AF) B(1)-induced toxicities, the effects of these flavonoids on the genotoxicities and oxidation of benzo[a]pyrene and AFB(1) were studied in C57BL/6J mice. Baicalein and wogonin reduced benzo[a]pyrene and AFB(1) genotoxicities as monitored by the umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. Baicalein added in vitro decreased liver microsomal benzo[a]pyrene hydroxylation (AHH) activity with an ic(50) of 33.9 +/- 1.4 microM at 100 microM benzo[a]pyrene. Baicalein also inhibited AFQ(1) and AFB(1)-epoxide formation from AFB(1) (50 microM) oxidation (AFO) with ic(50) values of 22.8 +/- 1.4 and 5.3 +/- 0.8 microM, respectively. However, the in vitro inhibitory effects of wogonin on AHH and AFO activities in liver microsomes were less than those of baicalein as inhibition by 500 microM wogonin was only about 51-65%. Treatment of mice with liquid diets containing 5 mM baicalein and wogonin resulted in 22 and 49% decreases in hepatic AHH activities, respectively. Baicalein treatment resulted in 39 and 32% decreases in AFQ(1) and AFB(1)-epoxide formation from liver microsomal AFO, respectively. Wogonin treatment resulted in 39 and 47% decreases in AFQ(1) and AFB(1)-epoxide formation, respectively. A 1-week pretreatment with wogonin significantly decreased hepatic DNA adduct formation in mice treated with 200 mg/kg of benzo[a]pyrene via gastrogavage. These in vitro and in vivo effects suggested that baicalein and wogonin might have beneficial effects against benzo[a]pyrene- and AFB(1)-induced hepatic toxicities and that wogonin had a stronger protective effect in vivo.

Binding of dimemorfan to sigma-1 receptor and its anticonvulsant and locomotor effects in mice, compared with dextromethorphan and dextrorphan.

Dextromethorphan ((+)-3-methoxy-N-methylmorphinan, DM) has been shown to have both anticonvulsant and neuroprotective effects. The mechanisms of these CNS effects of DM have been suggested to be associated with the low-affinity, noncompetitive, N-methyl-d-aspartate (NMDA) antagonism of DM and/or the high-affinity DM/sigma receptors. DM is largely O-demethylated into the phencyclidine (PCP)-like compound dextrorphan (DR), which may limit its therapeutic use by producing PCP-like adverse effects, such as hyperlocomotion. Dimemorfan ((+)-3-methyl-N-methylmorphinan, DF), an analog of DM, which has been safely used as an antitussive for more than 20 years, is also known not to form DR. This study therefore characterized the binding of DF to the sigma receptors and NMDA-linked PCP sites and examined the anticonvulsant as well as locomotor effects of DF in mice in comparison with those of DM and DR. We found that DF, DM, and DR were relative high-affinity ligands at sigma-1 receptors (Ki=151, 205, 144 nM, respectively) while all of them were with low affinity at sigma-2 receptors (Ki=4-11 microM). Only DR exhibited moderate affinity for PCP sites (Ki=0.9 microM), whereas DF (Ki=17 microM) and DM (Ki=7 microM) were much less active. DF, DM and DR produced prominent anticonvulsant effects in mice as measured by the supramaximal electroshock test with comparable potency (ED50 approximately 70 micromol/kg, i.p.). At the tested doses (20-260 micromol/kg, i.p.), DM and DR exhibited biphasic effects on the locomotor activity whereas DF produced a consistent dose-dependent decrease. These results revealed that, unlike DM and DR, DF did not cause a PCP-like hyperlocomotion adverse effect that is parallel with the PCP sites binding data. Furthermore, since they have equipotent anticonvulsant effects and similar binding affinities to sigma-1 receptors, the very low affinity of DF at PCP sites may suggest that acting on the PCP sites may not be the requisite for mediating the anticonvulsant activity of these DM analogs. With the history of safety and relative less adverse effects, DF appears to be worth further studying on its CNS effects other than the antitussive effect.

Dehydroevodiamine attenuates beta-amyloid peptide-induced amnesia in mice.

Dehydroevodiamine has been reported to have anticholinesterase activity and an anti-amnesic effect. This study examined the effects of dehydroevodiamine on scopolamine- and beta-amyloid peptide-(25--35)-induced amnesia in mice, using a step-through passive avoidance test. Similarly to the cholinesterase inhibitor, physostigmine (0.03--0.3 mg/kg, i.p.), dehydroevodiamine (0.75--12.0 mg/kg, i.p.) administered 30 min before the training trial, immediately after the training trial, and 30 min before the retention test significantly improved scopolamine- and beta-amyloid peptide-(25--35)-induced amnesia. In beta-amyloid peptide-(25--35)-induced amnesia, the rank order of anti-amnesic potency in these three administration schedules for dehydroevodiamine was different from that for physostigmine. Furthermore, dehydroevodiamine was more potent to improve beta-amyloid peptide-(25--35)-induced amnesia than scopolamine-induced amnesia when administered before the training trial. These results suggested that dehydroevodiamine may have an action other than that of an anticholinesterase and may be a novel and effective ligand for improvement of beta-amyloid type amnesia.

The mechanism of the vasodilator effect of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa.

The mechanisms underlying the rutaecarpine-induced vasodilatation were studied using isolated rat mesenteric arterial ring segments. The results showed that rutaecarpine (0.1 microM to 0.1 mM) produced a dose-dependent vasorelaxing response in our preparations, which were precontracted with phenylephrine. This vasodilator effect was significantly attenuated by removal of the endothelium, treatment with L-NG-nitro-arginine, a nitric oxide synthase inhibitor, and methylene blue, a guanylyl cyclase inhibitor, but not by treatment with atropine, triprolidine and yohimbine. Rutaecarpine pretreatment (1 microM to 0.1 mM) reduced both the phasic (fast) and tonic (slow) phases of phenylephrine-induced contractions, suggesting that a reduction in intracellular calcium might be involved. It is thus concluded that while the vasorelaxing effect of rutaecarpine appeared to be endothelium-dependent and to involve nitric oxide and guanylyl cyclase, neither muscarinic receptors, histamine H1 receptors nor alpha 2-adrenoceptors are involved. Moreover, a direct effect on the vascular smooth muscle cell, possibly through a reduction in intracellular Ca2+, can not be excluded.

Vasorelaxant effect of harman.

The in vivo cardiovascular effect and in vitro vasorelaxant effect of harman, one of harmala alkaloids isolated from Peganum harmala, were examined in this study. Harman (1-10 mg/kg, i.v.) dose-dependently produced transient hypotension and long-lasting bradycardia in pentobarbital-anesthetized rats, which were attenuated by N(G)-nitro-L-arginine pretreatment. In isolated rat endothelium-intact thoracic aortic rings, harman concentration dependently relaxed phenylepherine-induced contractions with an IC(50) value around 9 microM. This vasorelaxant effect was attenuated by endothelium removal or N(omega)-nitro-L-arginine methyl ester pretreatment, but not by tetraethylammonium or indomethacin pretreatment. In cultured rat aortic endothelial cells, harman (1-100 microM) concentration dependently increased nitric oxide (NO) release, which was dependent on the presence of external Ca(2+). Harman pretreatment (3-30 microM) also concentration dependently inhibited the contractions induced by phenylephrine, 5-hydroxytryptamine (5-HT), and KCl in endothelium-denuded aortic rings in a non-competitive manner. In addition, harman pretreatment reduced both phasic and tonic phases of phenylephrine-induced contractions. Receptor binding assays further indicated that harman (K(i) values around 5-141 microM) interacted with the cardiac alpha(1)-adrenoceptors, brain 5-HT(2) receptors, and cardiac 1, 4-dihydropyridine binding site of L-type Ca(2+) channels. Therefore, the present results suggested that the vasorelaxant effect of harman was attributed to its actions on the endothelial cells to release NO and on the vascular smooth muscles to inhibit the contractions induced by the activation of receptor-linked and voltage-dependent Ca(2+) channels. The vasorelaxant effect may be involved in the hypotensive effect of harman.

Effects of baicalein and wogonin on drug-metabolizing enzymes in C57BL/6J mice.

Effects of baicalein and wogonin, the major flavonoids of Scutellariae radix, on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. One-week treatment of mice with a liquid diet containing 5 mM baicalein resulted in 29%, 14%, 36%, 28%, and 46% decreases of hepatic benzo(a)pyrene hydroxylation (AHH), benzphetamine N-demethylation (BDM), N-nitrosodimethylamine N-demethylation (NDM), nifedipine oxidation (NFO), and erythromycin N-demethylation (EMDM) activities, respectively. Treatment with a liquid diet containing 5 mM wogonin resulted in 43%, 22%, 21%, 24%, and 35% decreases of hepatic AHH, BDM, NDM, NFO, and EMDM activities, respectively. However, hepatic 7-methoxyresorufin O-demethylation (MROD) activity was increased and decreased by baicalein- and wogonin-treatments, respectively. Similar modulation was observed with caffeine 3-demethylation (CDM) activity. Immunoblot analysis showed that the levels of hepatic CYP2E1 and CYP3A proteins were decreased by both baicalein- and wogonin-treatments. Hepatic CYP1A2 protein level was increased by baicalein but decreased by wogonin. In extrahepatic tissues, renal AHH activity was decreased by wogonin whereas pulmonary AHH, 7-ethoxyresorufin O-deethylation (EROD), and MROD activities were increased by both flavonoids. Both baicalein and wogonin strongly increased CYP1A protein level in mouse lung. Hepatic and renal UGT activities toward p-nitrophenol were suppressed by baicalein- and wogonin-treatments. However, cytosolic GST activity was not affected by flavonoids. These results suggest that ingestion of baicalein or wogonin can modulate drug-metabolizing enzymes and the modulation shows tissue specificity.

In vitro and in vivo effects of naringin on cytochrome P450-dependent monooxygenase in mouse liver.

In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60%. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM. Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38%. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.

Pharmacokinetics of glycyrrhizin after intravenous administration to rats.

A simple high-performance liquid chromatographic method was developed to study the pharmacokinetics of glycyrrhizin in the rat after bolus intravenous administration at a dose of 20, 50, or 100 mg/kg. The decline in the concentration of glycyrrhizin in plasma was generally biexponential at each dose, but the terminal disposition became much slower as the dose was increased. A greater-than-proportional increase in the glycyrrhizin concentration in plasma was observed with an increase in the dose, a result suggesting a dose-dependent glycyrrhizin disposition. The disposition of drug in plasma at each dose fitted well to a two-compartment pharmacokinetic model. The apparent total body clearance decreased significantly with increases in the dose. On the other hand, the apparent distribution volume after intravenous administration was unaffected by the dose. The results indicate that the pharmacokinetics of glycyrrhizin is nonlinear.

Anti-diarrheal effect of water extract of Evodiae fructus in mice.

Our previous study showed that Evodiae fructus (the dried, unripe fruit of Evodia rutaecarpa) has an inhibitory effect on the intestinal transit (anti-transit effect) in mice. In the present study, a water extract of Evodiae fructus was used to examine its effect on castor oil-induced diarrhea and to compare with its anti-transit effect in mice. The results indicated that Evodiae fructus had both anti-transit and anti-diarrheal effects with comparable ID(50) (the dose for 50% inhibition) values of 54+/-7 and 76+/-17 mg/kg. The time-courses of Evodiae fructus pretreatment for both anti-transit and anti-diarrheal effects were very similar. Because no significant influences of both nitric oxide (NO) precursor L-arginine (600 mg/kg, i.p.) and NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (25 mg/kg, i.p.) pretreatment, the NO system was not involved in both the anti-transit and anti-diarrheal effects of Evodiae fructus. Like Evodiae fructus, a muscarinic acetylcholine receptor antagonist atropine inhibited castor oil-induced increase in fecal weight and loss of body weight. However, the potencies or time-courses of atropine pretreatment for both anti-transit and anti-diarrheal effects were different. Furthermore, the anti-diarrheal effect of atropine was independent of its anti-transit effect at the lower dose (0.5 mg/kg, i.p.). Therefore, the action of Evodiae fructus appeared to be something different from atropine, suggesting that an action other than the anti-muscarinic action, as previously proposed for Evodiae fructus, may be involved.

Anticonvulsant effect of water extract of Scutellariae radix in mice.

Since a previous study indicated that the water extract of Scutellariae radix (SR) had high affinity for the benzodiazepine (BDZ) binding site of GABA(A) receptors, the present study examined whether SR water extract has an anticonvulsant effect in vivo and an enhancing effect on gamma-amino-n-butyric acid (GABA)-stimulated uptake of 36Cl(-) in cortex preparation in vitro in mice. The results showed that SR water extract had little effect on pentylenetetrazol (PTZ, 85 mg/kg, s.c.)-induced clonic seizures but significantly inhibited maximal electroshock-induced tonic seizures with an ED(50) of 3.6 g/kg. The BDZ agonist chlordiazepoxide (10 mg/kg, i.p.) had anticonvulsant activity on both types of seizures. In 36Cl(-) uptake assay, SR water extract (1-500 microg/ml) had no significant effect on 25 microM GABA-stimulated 36Cl(-) uptake, whereas chlordiazepoxide (10 microM) increased the 36Cl(-) uptake to 125% of control. Therefore, the present results showed for the first time that SR water extract had anticonvulsant activity against maximal electroshock-induced tonic seizures, and suggested that this anticonvulsant effect might be not via the activation of the BDZ binding site of GABA(A) receptors, but probably via the prevention of seizure spread.

Spasmolytic effect of water extract of Stemonae radix on the guinea-pig tracheal smooth muscle in vitro.

The present study examined the relaxing effect of a water extract of Baibu (Stemonae radix, the root tuber of Semona sessilifolia (Miq.) Franch. et Sav.) on carbachol-, histamine- and KCl-induced contractions of the guinea-pig isolated tracheal preparations. The results showed that Baibu (1-50 mg/ml) concentration-dependently relaxed the tracheal preparations contracted by these spasmogens with an IC50 value (mg/ml) of 2.0 +/- 0.1 for carbachol, 41.2 +/- 0.8 for histamine and 18.6 +/- 0.9 for KCl. The effect of Baibu was not affected by the pretreatment with a beta-adrenoceptor antagonist propranolol (10(-6) M), indicating that Baibu's effect was not due to an activation on beta-adrenoceptors. Baibu shifted the concentration-response curve of carbachol to the right in a parallel manner without changing the maximal response, having a pA2 value of 0.16 +/- 0.07 mg/ml (equivalent to a KB = 0.70 +/- 0.11 mg/ml). This indicates a competitive antagonism at the muscarinic receptors. Receptor binding assay indicated that Baibu interacted with the muscarinic receptors (Ki = 0.51 +/- 0.12 mg/ml) and the dihydropyridine (DHP) binding site of L-type Ca2+ channels (Ki = 8.0 +/- 1.9 mg/ml), but not with the histamine H1 receptors. Therefore, the present study demonstrates that Baibu contains the principle(s) acting on the muscarinic receptors and DHP binding sites, which contribute its relaxation effect on the airway smooth muscles.

Central inhibitory effects of water extract of Acori graminei rhizoma in mice.

The present study evaluated in mice the central inhibitory effects of a water extract of shichangpu (Acori graminei rhizoma (AGR), the dry rhizome of Acorus gramineus Soland. (Araceae)). AGR (0.5-5.0 g/kg) dose-dependently decreased the locomotor activity and increased the pentobarbital-induced sleeping time, but had no significant effect on the treadmill performance. AGR also dose-dependently inhibited the intensity of apomorphine-induced stereotypic behavior. At the highest dose (5.0 g/kg), AGR had a weak anticonvulsant effect on the pentylenetetrazol-induced seizures. Receptor binding assays showed that AGR competed with [3H]SCH-23390 and [3H]YM-09151-2 for specific binding to striatal dopamine D1 and D2 receptors with Ki values of 5.6 and 4.2 mg/ml, respectively. AGR also competed with [3H]muscimol for specific binding to the gamma-aminobutyric acid (GABA) binding site of cortex GABA(A) receptors with a Ki value of 0.31 mg/ml. It also increased the specific binding of [3H]flunitrazepam to the benzodiazepine binding site of the GABA(A) receptors, suggesting a GABA agonist-like action. These results suggested that the central inhibitory effects of AGR were probably effected through an action on the central dopamine receptors and GABA(A) receptors. The principle of AGR acting at these ligand binding sites was not alpha-asarone, one of the important principles of AGR, since that alpha-asarone (10(-6)-10(-4) M) had no significant interactions with these binding sites.

Agonist regulation of beta-adrenergic receptors: immunoblotting and indirect immunofluorescence reveal agonist-induced lateral sequestration and loss of binding.

The binding capacity and sub-cellular distribution of beta 2 adrenergic receptors was explored in rat osteosarcoma cells (ROS 17/2.8) following short-term (20 min) and long-term (24 hr) agonist treatment. ROS cells express about 27,000 beta 2 adrenergic receptors per cell, nearly 90% of which appear plasma membrane-associated by radioligand binding analysis. When cells were treated with isoproterenol for 20 min, lysed, and the lysate centrifuged over a gradient of sucrose, beta-adrenergic receptors in the plasma membrane fraction were found to decline from approximately 90% to 50%, while those in a lower-density "light" vesicle fraction increased from approximately 10% to 50%. Immunoblotting with specific antisera against the beta 2 adrenergic receptor revealed, in sharp contrast, that 60% of receptor was distributed in the plasma membrane-enriched fraction, 40% in the light vesicle fraction of untreated cells. A 20-min exposure to agonist caused the 60:40 distribution of immunoreactive receptor between the two fractions (plasma membrane:light vesicle) to shift to 35:65. The distribution of immunoreactive G beta 1-subunit (36,000-M(r)) between the two fractions was 80:20 and not influenced by exposure of cells to agonist. A 24 hr-exposure to agonist changed markedly the distribution of immunoreactive receptor from 60:40 to 90:10 (plasma membrane: light vesicle). Receptor content as determined by radioligand binding, in contrast, was nondetectable in either fraction prepared from ROS cells stimulated by agonist for 24 hr. Immunoblotting of post-nuclear, supernatant fractions of whole-cell lysates revealed no change in receptor content after 24 hr of agonist treatment. Furthermore, SDS-polyacrylamide gel electrophoresis and immunoblotting revealed no prominent proteolytic degradation of receptor in response to agonist stimulation at 20 min or 24 hr. Indirect immunofluorescence of beta adrenergic receptors in fixed, intact ROS cells probed only cell surface-associated receptor and yielded equivalent epifluorescence signals for untreated cells and cells treated with isoproterenol for either 20 min, or 24 hr. The immunological results confirm the phenomenon of agonist-induced receptor sequestration, but reveal several new insights: (i) beta adrenergic receptor protein content and subcellular distribution may not be accurately reflected by radioligand binding; (ii) receptor down-regulation (loss of binding) after 24 hr exposure to agonist cannot be explained by enhanced receptor degradation; (iii) the cell surface complement of receptor is not altered at 20 min or 24 hr following stimulation of cells with agonist; and (iv) lateral sequestration of receptors to separate domains of the cell membrane occurs when ROS 17/2.8 cells are exposed to beta-agonist for a short time (20 min).