Mediation of oxidative stress in hypothalamic ghrelin-associated appetite control in rats treated with phenylpropanolamine.

Phenylpropanolamine (PPA)-induced appetite control is associated with oxidative stress in the hypothalamus. This study explored whether hypothalamic antioxidants participated in hypothalamic ghrelin system-associated appetite control in PPA-treated rats. Rats were given PPA daily for 4 days, and changes in food intake and the expression of neuropeptide Y (NPY), the cocaine- and amphetamine-regulated transcript (CART), superoxide dismutase, catalase, ghrelin, acyl ghrelin (AG), ghrelin O-acyltransferase (GOAT) and the ghrelin receptor (GHSR1a) were examined and compared. Results showed that both food intake and the expression of NPY and ghrelin/AG/GOAT/GHSR1a decreased in response to PPA treatment with maximum decrease on Day 2 of the treatment. In contrast, the expression of antioxidants and CART increased, with the maximum increase on Day 2, with the expression opposite to that of NPY and ghrelin. A cerebral infusion of either a GHSR1a antagonist or reactive oxygen species scavenger modulated feeding behavior and NPY, CART, antioxidants and ghrelin system expression, showing the involvement of ghrelin signaling and oxidative stress in regulating PPA-mediated appetite control. We suggest that hypothalamic ghrelin signaling system, with the help of antioxidants, may participate in NPY/CART-mediated appetite control in PPA-treated rats.

Central dopamine action modulates neuropeptide-controlled appetite via the hypothalamic PI3K/NF-κB-dependent mechanism.

Hypothalamic neuropeptides, including neuropeptide Y (NPY) and proopiomelanocortin (POMC), have been found to control the appetite-suppressing effect of amphetamine (AMPH). In this study, we have examined whether dopamine receptor (DAR), phosphatidylinositol 3-kinase (PI3K) and nuclear factor-kappaB (NF-κB) are involved in AMPH's action. We administered AMPH to rats once a day for 4 days and assessed and compared changes in hypothalamic NPY, melanocortin receptor 4 (MC4R), PI3K, pAkt and NF-κB expression. We found that the inhibition of DAR increased NPY, but decreased MC4R, PI3K and NF-κB expression, compared with AMPH-treated rats. Moreover, MC4R, PI3K, pAkt and NF-κB increased with the maximum response on Day 2, which was consistent with the response of feeding behavior, but was opposite to the expression of NPY. Furthermore, we found that the intracerebroventricular infusion of the PI3K inhibitor or NF-κB antisense could attenuate AMPH-induced anorexia, and partially reverse the expression of NPY, MC4R, PI3K, Akt and NF-κB back toward a normal level. We, therefore, suggest that DAR-PI3K-NF-κB signaling in the hypothalamus plays functional roles in the modulation of NPY and POMC neurotransmissions and in the control of AMPH-evoked appetite suppression.

Role of lipocalin 2 and its complex with matrix metalloproteinase-9 in oral cancer.

OBJECTIVES: Recent evidence demonstrated that lipocalin (LCN)2 is induced in many types of human cancer, while the detection of its complex with matrix metalloproteinase (MMP)-9 is correlated with the cancer disease status. We attempted to evaluate plasma expressions of LCN2, MMP-9, and their complex (LCN2/MMP-9) during the diagnostic work-up of patients with oral squamous cell carcinoma (OSCC) and investigated their correlations with disease progression. METHODS: In total, 195 patients with OSCC and 81 healthy controls were recruited. Expression levels of LCN2, MMP-9, and LCN2/MMP-9 were determined with immunoenzymatic assays. RESULTS: Patients with OSCC exhibited significantly higher levels of LCN2, MMP-9, and LCN2/MMP-9 compared with healthy controls (LCN2: P < 0.001; MMP-9: P < 0.001; LCN2/MMP-9: P < 0.01). Plasma levels of LCN2, MMP-9, and LCN2/MMP-9 in patients with OSCC were significantly correlated with each other and were associated with more-advanced clinical stages (P < 0.05) and/or a larger tumor size (P < 0.05), but were not associated with positive lymph-node metastasis or distal metastasis. CONCLUSION: Our results suggest that plasma levels of LCN2 and the LCN2/MMP-9 complex may be useful in non-invasively monitoring OSCC progression, while supporting their potential role as biomarkers of oral cancer disease status.

Effects of silymarin on the resolution of liver fibrosis induced by carbon tetrachloride in rats.

Silymarin, a standardized extract of the milk thistle (Silybum marianum), has a long tradition as a herbal remedy, and was introduced as a hepatoprotective agent a few years ago. However, the therapeutic effects of silymarin remain undefined. Carbon tetrachloride (CCl4) is a xenobiotic used extensively to induce oxidative stress and is one of the most widely used hepatic toxins for experimental induction of liver fibrosis in the laboratory. In this study, we investigated the restoration of the CCl4-induced hepatic fibrosis by high dose of silymarin in rats. After treatment with oil (as normal group; n = 6) or CCl4 [as model (n = 7) and therapeutic (n = 7) groups] by intragastric delivery for 8 weeks for the induction of liver fibrosis, the rats in the normal and model group were administered orally normal saline four times a week for 3 weeks whilst the therapeutic group received silymarin (200 mg/kg). The histopathological changes were observed with Masson staining. The results showed that the restoration of the CCl4-induced damage of liver fibrosis in the therapeutic group was significantly increased as compared to that in the model group. Moreover, silymarin significantly decreased the elevation of aspartate aminotransferase (AST), alanine aminotransferase, and alkaline phosphatase in serum, and also reversed the altered expressions of alpha-smooth muscle actin in liver tissue. Therefore, these findings indicated that silymarin may have the potential to increase the resolution of the CCl4-induced liver fibrosis in rats.

Luteolin induces apoptosis in oral squamous cancer cells.

Oral squamous cell carcinoma is the most common malignancy of the oral cavity, and treatment approaches are inadequate. Luteolin, a natural flavonoid compound, has been shown to have anti-tumorigenic properties on various types of tumors. Therefore, we hypothesized that luteolin has anti-tumorigenic properties for oral squamous cell carcinoma, and may provide effective chemotherapy. Results revealed that luteolin reduced the viability of SCC-4 cells and induced apoptosis by decreasing the expression of cyclin-dependent kinase (CDKs), cyclins, and phosphor- retinoblastoma (p-Rb) anti-apoptotic protein, but increased the expression of pro-apoptotic proteins and activated caspase 9 and 3, with a concomitant increase in the levels of cleaved poly-ADP-ribose polymerase (PARP). Combination treatment of luteolin with paclitaxel enhanced the cytotoxic effect of paclitaxel in SCC-4 cells, and continuous administration of luteolin suppressed the growth of xenograft tumors in nude mice. These results suggest that luteolin could be an effective chemotherapeutic agent for the treatment of oral squamous cell carcinoma.

Increased plasminogen activator inhibitor-1/tissue type plasminogen activator ratio in oral submucous fibrosis.

OBJECTIVE: Plasminogen activators and their inhibitors are thought to be key participants in the balance of proteolytic and antiproteolytic activities that regulate extracellular matrix (ECM) turnover. However, little is known about the expression of plasminogen/plasmin system at the site of oral submucous fibrosis (OSF). METHODS: We compared the activities of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) between fibroblasts derived from normal buccal mucosa and OSF by using an enzyme-linked immunosorbent assay. Furthermore, arecoline, a major areca nut alkaloid, was challenged with normal buccal mucosal fibroblasts (BMFs) to elucidate whether the activities of t-PA and PAI-1 could be affected by arecoline. RESULTS: Both t-PA and PAI-1 were found to be increased in OSF than in BMFs (P < 0.01). In addition, there was a statistically significant difference in PAI-1/t-PA ratio between OSF and BMF (P < 0.01). The addition of arecoline upregulated not only PAI-1, but also t-PA in BMFs (P < 0.05). In addition, the ratio between PAI-1 and t-PA was found to be significantly increased by a linear regression assay (P < 0.01). CONCLUSION: These results suggest that OSF caused by areca quid chewing may be the result of an imbalance in the plasminogen/plasmin system, the net result of which is increased deposition of ECM.

Silibinin inhibits invasion of oral cancer cells by suppressing the MAPK pathway.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity. Here, we provide molecular evidence associated with the anti-metastatic effect of silibinin by showing a marked inhibition of the invasion and motility of SCC-4 tongue cancer cells, with 89% and 66.4% of inhibition, respectively, by 100 microM of silibinin. This effect was associated with a reduced expression of MMP-2 and u-PA, together with an enhanced expression of TIMP-2 and PAI-1. Silibinin also exerted an inhibitory effect on the phosphorylation of ERK1/2. Additionally, pre-treatment of SCC-4 cancer cells with 10 and 20 microM of U0126, a specific MEK inhibitor, resulted in a reduced expression of MMP-2 (18.7 and 51.4%) and u-PA (19.2 and 48.9%) concomitantly with a marked inhibition of cell invasion (13.7 and 45.7%). Finally, silibinin was evidenced by its inhibition of the metastasis of Lewis lung carcinoma (LLC) cells in vivo. These results suggested that silibinin can reduce the invasion and metastasis of tumor cells, and such a characteristic may be of great value in the development of a potential cancer therapy.

Regulation of interleukin-6 expression by arecoline in human buccal mucosal fibroblasts is related to intracellular glutathione levels.

OBJECTIVES: Cytokines play an important role in regulating fibroblast function and is likely to play a key role in regulating the initiation and progression of scarring in any fibrotic disease. Interleukin-6 (IL-6) has been implicated in the development of a variety of fibrotic diseases. The aim of this study was to compare IL-6 expression in fibroblasts cultured from normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce IL-6 expression. METHODS: mRNA level of IL-6 in fibroblasts from OSF was compared with normal buccal mucosa. The effects of arecoline, the major areca nut alkaloid, on IL-6 expression in normal human buccal mucosa fibroblasts (BMFs) were measured in vitro. mRNA was quantified with AlphaImager 2000. To determine whether glutathione (GSH) levels were important in the induction of IL-6 by arecoline, we pretreated cells with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or with buthionine sulfoximine (BSO) to deplete GSH. RESULTS: Fibroblasts derived from OSF exhibited higher IL-6 gene expression than BMF in mRNA levels (P < 0.05). The exposure of quiescent BMF to arecoline resulted in the elevation of IL-6 mRNA expression in a dose-dependent manner (P < 0.05). IL-6 gene regulated by arecoline correlated with intracellular GSH levels in BMF. Arecoline at a concentration of 129 muM induced about 2.7-fold IL-6 mRNA levels over the 6-h incubation period. However, BSO enhanced the IL-6 mRNA levels by 3.9-fold (P < 0.05). In addition, OTZ was found to marginally reduce the arecoline-induced IL-6 expression by about 1.7-fold (P < 0.05). CONCLUSIONS: Taken together, these results suggest that IL-6 expression is significantly upregulated in OSF fibroblasts in areca quid chewers and arecoline may be responsible for the enhanced IL-6 expression. In addition, the regulation of IL-6 expression induced by arecoline is critically dependent on the intracellular GSH concentrations.

Alternations in quantities and activities of erythrocyte cytosolic carbonic anhydrase isoenzymes in glucose-6-phosphate dehydrogenase-deficient individuals.

BACKGROUND: This study was designed to evaluate the quantitative and activity alterations of cytosolic carbonic anhydrase (CA) isoenzymes in the erythrocytes of glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals. METHODS: Western Blot and CA esterase activity analysis were employed to measure cytosolic erythrocyte CA isoenzymes. RESULTS: The total CA activities were analyzed from erythrocytes of 30 healthy and 30 G6PD-deficient individuals. The mean values with standard error (SE) were 22.9+/-1.69 U/gHb and 27.2+/-2.1 U/gHb (P<0.01), respectively. The ratio of CAI/CAII of G6PD-deficient individuals (1.28+/-0.06) was significantly lower than that of the normal subjects (3.79+/-0.18) (P<0.001). Furthermore, the concentration of CAIII in G6PD-deficient individuals was significantly lower than that of the normal subjects (P<0.001) and there were significant correlations between the concentration of CAI, CAII, CAIII, and ratio of CAI/CAII, and the activity concentration of G6PD. CONCLUSIONS: Different carbonic anhydrase isoenzymes may serve different roles in the G6PD-deficient erythrocyte. CAI could be used as an indicator for hemolytic anemia. CAII is able to compensate for the functions of CAI and increased expression of CAII will promote oxidative damage. CAIII can provide the G6PD-deficient persons with some extent of protection against oxidative damage.

Regulation of matrix metalloproteinase-2 production by cytokines and pharmacological agents in human pulp cell cultures.

Type IV matrix metalloproteinases (MMPs) are members of the family of MMPs and are thought to play an important role in degradation of extracellular components. Human pulp cells can secrete and produce these enzymes. Recent evidence shows that MMPs may play a role in pulpal inflammation. To date little is known regarding the regulation of MMPs in human pulp cell cultures. The purpose of this study was to determine the effects of cytokines (interleukin-1 and transforming growth factor-beta (TGF-beta), protein synthesis inhibitor cycloheximide (CD), and protein kinase C inhibitors (H7 and Go6976) on the secretion and production of MMPs by human pulp cell cultures using gelatin zymography. The main gelatinase secreted by human pulp cells migrated at 72 kDa and represented MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. After an 8-day culture period TGF-beta, CD, H7, and Go6976 were found to depress MMP-2 production. The inhibition decreased in an order of CD > H7 > TGF-beta > Go6976. IL-1 was found to elevate MMP-2 production. Human pulp cells, however treated with either cytokines or pharmacological agents had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. These observations suggest that the cytokines and pharmacological agents can regulate MMP-2 produced by human pulp cells. Inflammatory cytokines stimulate the production of elevated levels of MMP-2 and MMP-2 might play a role in pulpal inflammation. In addition agents that target protein synthesis or the protein kinase C pathway in human pulp cells inhibit MMP-2 production, and such inhibition may contribute to the pathogenesis of pulpal inflammation. Such inhibition might contribute to therapeutic efficacy.

Gaseous nitric oxide-induced 8-nitroguanine formation in human lung fibroblast cells and cell-free DNA.

A time- and dose-dependent increase in 8-nitroguanine (8-NO(2)-G) was observed in human lung fibroblast cells (MRC-5) after treatment with gaseous NO-saturated buffer. It was also found that treatment with the inhibitor of inducible nitric oxide synthase (iNOS), N(G)-nitro-l-arginine methyl ester, significantly reduced the 8-NO(2)-G level in the gaseous NO-saturated buffer-treated MRC-5 cells. These results provide evidence indicating that NO gas causes DNA damage in mammalian cells, which involves the activation of iNOS and the subsequent generation of endogenous NO. On the other hand, a time- and dose-dependent increase in 8-NO(2)-G was also observed while DNA (isolated from MRC-5 cells) was incubated with gaseous NO-saturated buffer. These results suggest that part of the 8-NO(2)-G formation was due to direct modification of gaseous NO on DNA. Furthermore, an increase in nitrite concentration was found in both cell-free and MRC-5 cell-conditioned medium treated with gaseous NO-saturated buffer. Collectively, gaseous NO induced DNA damage by forming 8-NO(2)-G, a modification performed directly by the treated gaseous NO and indirectly by the following induction of endogenous NO. This effect might be an important pathway in genotoxicity of nitric oxides, and 8-NO(2)-G could act as a specific marker for DNA damage induced by gaseous NO, a common contaminatant in air pollution and cigarette smoke.

Alteration in the expression of protein kinase C isoforms in human hepatocellular carcinoma.

This study was designed to investigate the alterations of individual protein kinase C (PKC) isoforms in human liver cancer. Surgical specimens of hepatocellular carcinoma and adjacent normal tissues were extracted into cytosolic and membranous fractions. The level of membrane-bound PKCalpha in the cancer tissue was significantly lower than that in the adjacent normal tissue and consistent with the change in PKC activity. In addition, there was a significant negative correlation between PKCalpha and tumor size. In both cytosolic and membrane fractions, levels of PKCdelta and PKCzeta was significantly higher in the cancer tissue than those in the adjacent normal liver tissue. The alterations in the PKC isoforms signify their roles in the hyperproliferation in liver cancer.

Significant differences in serum activities of matrix metalloproteinase-2 and -9 between HCV- and HBV-infected patients and carriers.

To examine the possible involvement of MMP-9 and -2 in the development of liver diseases caused by HCV or HBV infection, serum activities of both enzymes were studied by zymograph. Eight groups of subjects (60 for each) were examined in the study: healthy control, patients with hepatoma, liver cirrhosis, chronic hepatitis B or chronic hepatitis C, and carriers positive for HBsAg, both HBsAg and HBeAg, or anti-HCV. The results showed significant changes in the MMP-9 and -2 activities in the carriers. The presence of HBeAg was accompanied by a highest activity of MMP-2 and an inversely correlated (r=-0.578, P=<0.001), lowest activity of MMP-9 among all groups. For those with active liver diseases, MMPs activities were fluctuated at each stage of pathological symptoms. Chronic hepatitis B and C patients had significant different serum MMP-2 and -9 activities. These findings imply an influence on the balance of MMPs system by the existence of virus that might influence the following progression of liver disease, and a distinction between the pathological mechanisms of HCV and HBV. Since the serum MMPs activities were significantly varied between each stage of liver disease, an individual profile of these parameters might serve as an easy accessing serum marker to monitor the progression of liver disease.

Accuracy of ultrasonography in the diagnosis of peritonitis compared with the clinical impression of the surgeon.

HYPOTHESIS: Peritonitis is a well-known indication for surgery, but its preoperative cause usually is not established. We hypothesize that abdominal ultrasonography is superior to the clinical impression of the surgeon in detecting the cause of peritonitis. DESIGN: A prospective case series. SETTING: A major university hospital in Taiwan, Republic of China. PATIENTS AND METHODS: One hundred two patients with a diagnosis of peritonitis admitted to the Department of Emergency Medicine, National Taiwan University Hospital, Taipei, were included in this study. All 102 patients underwent an abdominal ultrasonographic examination; and the ultrasonographic findings of these patients were classified into 2 categories: positive findings and normal screening results. The accuracy of clinical impression in detecting the cause of peritonitis was compared with the accuracy of abdominal ultrasonography. RESULTS: Ultrasonography and clinical impression accurately diagnosed the peritonitis in 85 (83.3%) and 52 (51.0%) of the patients, respectively. The difference between ultrasonography and clinical impression in the diagnosis of peritonitis was significant (P<.001). Among 45 patients without a preoperative clinical diagnosis, a diagnosis was made by ultrasonography for 32 (71%) of them. There were a total of 98 patients with positive ultrasonographic findings, and 4 patients had normal screening results. Of the 98 patients with positive ultrasonographic findings undergoing surgery, all had abdominal pathological characteristics. The 4 patients with normal screening results received nonoperative treatment. CONCLUSIONS: Ultrasonography is a more sensitive technique than clinical judgment in diagnosing peritonitis. Ultrasonography may be a useful diagnosing modality in patients with peritonitis in whom the clinical cause is unclear.

Protein kinase C isoforms during the development of deciduomata in pregnant rats.

In this study, we determined the expression of protein kinase C (PKC) isoforms during pregnancy. At pregnant duration, PKC alpha was down-modulated in the deciduomata but not in the myometrium. Down-modulation was compatible with the increase in cell mitosis, which reached a maximum at 8-9 days. On the other hand, PKC zeta was not down-modulated. It was increased both in the cytosolic and particulate fractions of the deciduomata, and paralleled the frequency of decidual cell mitosis. The other PKC isoform of delta was also increased, but it was associated with the cell regression. Therefore, these findings confirmed that the variable expression of PKC isoforms in decidualizing tissue may be involved in the modulation of decidual cell growth.

Protein kinase C isoforms in the epidermal tissues of normal and postburn human skin.

Because the expression of the isoforms of protein kinase C (PKC) in human basal keratinocytes is not understood, the expression of PKC isoforms were screened in specimens of epidermal tissue from postburn skin and the normal locations for skin grafts in patients with second or higher degrees of flame injury. The expression of individual isoform was determined by Western blot technique. Only PKC alpha and zeta were detected in the epidermal tissues of normal and postburn skin and translocation occurred in PKC alpha. Patients without antibiotic treatment after flame injury had higher expressions of PKC alpha and zeta. These findings indicate that the mechanisms of cellular differentiation and growth in postburn epidermal tissue may be related to the expression and translocation of PKC alpha induced by intra- and extracellular stimulation. These changes in PKC alpha further activate the DAG/PKC signal transduction pathways.

Terpenoids of Syzygium formosanum.

A new natural product, 4-epifriedelin (1), and 12 known terpenoids have been isolated from the leaves of Syzygium formosanum. The known compounds include caryophyllene oxide, friedelin, canophyllal, glutinol, alpha-terpineol, phytol, betulinic acid, uvaol, lupeol, betulin, ursolic acid, and oleanolic acid. All of these compounds are reported for the first time from S. formosanum.

Protein kinase C isoforms during the development of deciduomata in pseudopregnant rats.

In this study, we determined the expression of protein kinase C (PKC) isoforms during trauma-induced decidualization. The findings revealed that at least five PKC isoforms (alpha, delta, zeta, iota and lambda) were present in both control and decidualized tissues. After trauma-stimulation, PKC alpha was down-modulated in the deciduomata but not in the myometrium. Down-modulation was compatible with the increase in cell mitosis which reached a maximum at 2-3 days. On the other hand, PKC zeta was not down-modulated. It was increased both in the deciduomata and myometrium, and paralleled the frequency of decidual cell mitosis. The PKC isoforms of delta, iota and lambda were also increased, but they were associated with the depression of cell mitosis. Therefore, these findings suggested that the variable expression of PKC isoforms in trauma-induced decidualizing tissue in pseudopregnant rats may be involved in the modulation of decidual cell growth.

Augmentation of protein kinase C activity and liver cell proliferation in lead nitrate-treated rats.

It is shown that lead alters calcium mediated cellular processes in several biological systems. Calcium enhances the activity of protein kinase C (PKC) which takes part in eliciting cell mitosis. In this study, the effects of lead nitrate on the activity of PKC enzyme were investigated in rat liver. The PKC activity was determined at 12, 24, 48, 72, 120, 168 hours after treatment with a single dose of lead nitrate in male Wistar rats. The results showed that the specific PKC activity of the purified particulate fraction was increased and reached a maximum at 24 hour, and lasted for 48 hours. This augmented activity of PKC was parallel with the increase of the lead level in the purified particulate fraction, although the protein levels of PKC alpha, PKC delta and PKC zeta were unchanged. Moreover, the frequency of mitotic cells also exhibited a significant increase, and like PKC activity, reached its maximum at 24 hour with accompany signs of liver enlargement. The results suggest that the PKC activation may be involved in promoting liver cell proliferation in lead nitrate-treated rats.