Facile determination of natural cannabinoids in cannabis products using a conventional fully porous particle column and isocratic high-performance liquid chromatography with diode-array detector

Forensic Toxicology -     

p38丝裂原活化蛋白激酶信号途径在鼠破骨细胞生成和骨吸收中的作用

目的研究p38丝裂原活化蛋白激酶(p38MAPK)信号途径在甲状旁腺素相关肽(PTHrP)诱导的破骨细胞生成和骨吸收中的作用。方法取小鼠骨髓细胞,在PTHrP(45ng/ml)的刺激下,在不同试验组中分别入0.1、1.0及10μmol/L的p38MAPK抑制剂Fr167653,继续培养6d。抗酒石酸染色,进行破骨细胞计数。在小鼠颅骨部位注射PTHrP建立骨吸收和高钙血症动物模型。每日给予p38MAPK抑制剂Fr16765330mg/kg,每日2次,X线片观察骨吸收面积,组织学检查计算单位面积内破骨细胞数目,采集血样观察全血内游离钙水平。结果PTHrP刺激下,大量破骨细胞生成(118.9±28.3)个/孔;加入0.1μmol/LFr167653可以部分抑制破骨细胞的生成(79.6±28.0)个/孔,加入10μmol/LFr167653几乎全抑制了破骨细胞生成(7.4±0.4)个/孔,每日给予Fr16765330mg/kg,每日2次,可以明显抑制骨吸收,表现为X线片上骨吸收面积减少,单位面积内破骨细胞数目减少,但是并不能有效地抑制高钙血症。结论抑制p38MAPK信号途径可以抑制破骨细胞的分化和局部骨吸收。     

Mechanism of superoxide anion production by hepatic sinusoidal endothelial cells and Kupffer cells during short‐term ethanol perfusion in the rat

Abstract: Background/Aims: The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O₂^·?) release into the hepatic sinusoids during short‐term exposure to ethanol. Methods: The rat liver was perfused continuously with ethanol (a substrate for alcohol dehydrogenase) or tert‐buthanol (not a substrate for alcohol dehydrogenase) for 20 min at a final concentration of 40 mM. In order to detect O₂^·? production, MCLA (2‐methyl‐6‐[p‐methoxyphenyl]‐3,7‐dihydroimidazo[1,2‐a]pyrazin‐3‐one), a Cypridina luciferin analogue, was simultaneously infused and MCLA‐enhanced chemiluminescence was measured. The effects of gadolinium chloride (GdCL₃) (a suppressor of Kupffer cells (KCs)), staurosporine (ST) (an inhibitor of serine–threonine kinases, including protein kinase C), diphenyleneiodonium chloride (DPI) (an inhibitor of NADPH oxidase), ibuprofen (IB) (an inhibitor of cyclooxygenase) and 4‐methylpyrazole (4MP) (an inhibitor of ethanol metabolism) on the ethanol‐induced chemiluminescence were also evaluated. Sites where O₂^·? could be released were determined by histochemical detection of nitro blue tetrazolium reduction. Results: Both ethanol and tert‐buthanol rapidly caused O₂^·? release. GdCL₃ suppressed the ethanol‐induced O₂^·? release by 61%. Staurosporine and DPI, but neither IB nor 4‐MP, also significantly inhibited the ethanol‐induced O₂^·? release. In the histochemical examination, ethanol‐stimulated liver showed blue formazan precipitate on both sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), whereas the GdCl₃‐pretreated liver had the precipitate only on SECs. Conclusions: This study shows that ethanol itself stimulates both SECs and KCs to release O₂^·? via activation of NADPH oxidase probably involving protein kinase C (PKC).     

The relationship between gallbladder disease and smoking and drinking habits in middle-aged Japanese

Background: Background: Few studies have investigated the association between smoking and ultrasonographically diagnosed gallbladder (GB) disease, and their results were uncertain. This study was conducted to examine the association between smoking and drinking and GB diseases. Methods: A total of 9947 subjects (age, 30–69 years; 4953 men and 4994 women) voluntarily received a paid medical check-up at our center in Yamanashi Prefecture in Japan. All of the subjects underwent abdominal ultrasonographic (US) examination, a demographic check, and a biochemical test, and answered a self-administered questionnaire asking about smoking habits and alcohol consumption. Of the 9947 subjects, 483 had gallstones, 819 had gallbladder polyps, and 169 were in a state of postcholecystectomy. We compared the findings in this group with the findings in 8417 people (4144 males and 4273 females) with normal gallbladder. Results: Multiple regression analysis among males showed that cigarette smoking was inversely related to GB polyps (odds ratio, [OR], 0.76; 95% confidence internal [CI], 0.59–0.98 and OR, 0.74; 95% CI, 0.56–0.98, respectively, for current and ex-smokers). Ex-smokers a showed positive association with the postcholecystectomy state (OR, 2.56; 95% CI, 1.18–5.52). Light drinkers showed an inverse relation to GB stones (OR, 0.69; 95% CI, 0.49–0.99), and heavy drinkers showed an inverse relation to GB polyps (OR, 0.68; 95% CI, 0.51–0.90). Current drinkers showed an inverse relation to the postcholecystectomy state (OR, 0.48; 95% CI, 0.28–0.83). Conclusions: Cigarette smoking was inversely related to gallbladder polyps in males and was positively related to the postcholecystectomy state. Drinking was inversely related to gallstones, GB polyps, and the postcholecystectomy state in males. Received: July 19, 2001 / Accepted: November 2, 2001     

Ozone production by amino acids contributes to killing of bacteria

Reactive oxygen species produced by phagocytosing neutrophils are essential for innate host defense against invading microbes. Previous observations revealed that antibody-catalyzed ozone formation by human neutrophils contributed to the killing of bacteria. In this study, we discovered that 4 amino acids themselves were able to catalyze the production of an oxidant with the chemical signature of ozone from singlet oxygen in the water-oxidation pathway, at comparable level to antibodies. The resultant oxidant with the chemical signature of ozone exhibited significant bactericidal activity in our distinct cell-free system and in human neutrophils. The results also suggest that an oxidant with the chemical signature of ozone produced by neutrophils might potentiate a host defense system, when the host is challenged by high doses of infectious agents. Our findings provide biological insights into the killing of bacteria by neutrophils.     

The origin of the parathyroid gland

It has long been held that the parathyroid glands and parathyroid hormone evolved with the emergence of the tetrapods, reflecting a need for new controls on calcium homeostasis in terrestrial, rather than aquatic, environments. Developmentally, the parathyroid gland is derived from the pharyngeal pouch endoderm, and studies in mice have shown that its formation is under the control of a key regulatory gene, Gcm-2. We have used a phylogenetic analysis of Gcm-2 to probe the evolutionary origins of the parathyroid gland. We show that in chicks, as in mice, Gcm-2 is expressed in the pharyngeal pouches and the forming parathyroid gland. We find that Gcm-2 is present not only in tetrapods but also in teleosts and chondrichthyans, and that in these species, Gcm-2 is expressed within the pharyngeal pouches and internal gill buds that derive from them in zebrafish (Danio rerio), a teleost, and dogfish (Scyliorhinus canicula), a chondrichthyan. We further demonstrate that Gcm-2 is required for the formation of the internal gill buds in zebrafish. We also have identified parathyroid hormone 1/2-encoding genes in fish and show that these genes are expressed by the gills. We further show that the gills express the calcium-sensing receptor, which is used in tetrapods to monitor serum calcium levels. These results indicate that the tetrapod parathyroid gland and the gills of fish are evolutionarily related structures, and that the parathyroid likely came into being as a result of the transformation of the gills during tetrapod evolution.     

Interleukin-17A promotes rheumatoid arthritis synoviocytes migration and invasion under hypoxia by increasing MMP2 and MMP9 expression through NF-κB/HIF-1α pathway

Both hypoxia and interleukin-17A (IL-17A) promote the migration and invasion of fibroblast-like synoviocytes (FLSs), which are critical for the pathogenesis of rheumatoid arthritis (RA). However, the biochemical pathways regulating IL-17A combined with hypoxia are not well defined. In this study, we found that co-stimulating RA-FLSs with IL-17A and hypoxia did not appear to promote the epithelial–mesenchymal transition (EMT), but did increase cell motility. We further showed that a proinvasive effect of IL-17A on FLSs under hypoxia might be through upregulation of matrix metalloproteinase 2 (MMP2) and MMP9. Moreover, IL-17A-induced expression of MMP2 and MMP9 under hypoxia was accompanied by increased activation of nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α). Knockdown or inhibition of HIF-1α and NF-κB by small interfering RNA or specific small molecule inhibitors blocked IL-17A-mediated and hypoxia-mediated MMP2 and MMP9 expression, cell migration, and invasion. In addition, the inhibition of NF-κB led to a marked decrease in the expression of HIF-1α, which indicated that IL-17A activated HIF-1α via the NF-κB pathway in hypoxia. Taken together, our observations suggest a synergetic effect of IL-17A and hypoxia that might contribute to the migration and invasion of RA-FLSs by upregulating the expression of MMP2 and MMP9 by activation of the NF-κB/HIF-1α pathway.     

Signaling for lymphangiogenesis via VEGFR-3 is required for the early events of metastasis

Metastasis to regional lymph nodes is an important and early event in many tumors. Vascular endothelial growth factor-C (VEGF-C), VEGF-D and their receptor VEGFR-3, play a role in tumor spread via the lymphatics, although the timing of their involvement is not understood. In contrast, VEGFR-2, activated by VEGF-A, VEGF-C and VEGF-D, is a mediator of angiogenesis and drives primary tumor growth. We demonstrate the critical role for VEGFR-3, but not VEGFR-2, in the early events of metastasis. In a tumor model exhibiting both VEGF-D-dependent angiogenesis and lymphangiogenesis, an antibody to VEGFR-2 (DC101) was capable of inhibiting angiogenesis (79 % reduction in PECAM + blood vessels) and growth (93 % reduction in tumor volume). However, unlike an anti-VEGFR-3 Mab (mF4-31C1), DC101 was not capable of eliminating either tumor lymphangiogenesis or lymphogenous metastasis (60 % reduction of lymph node metastasis by DC101 vs 95 % by mF4-31C1). Early excision of the primary tumors demonstrated that VEGF-D-mediated tumor spread precedes angiogenesis-induced growth. Small but highly metastatic primary human breast cancers had significantly higher lymphatic vessel density (23.1 vessels/mm²) than size-matched (11.7) or larger non-metastatic tumors (12.4) thus supporting the importance of lymphatic vessels, as opposed to angiogenesis-mediated primary tumor growth, for nodal metastasis. These results suggest that lymphangiogenesis via VEGF-D is more critical than angiogenesis for nodal metastasis.