[Clinicopathologic features of hepatocellular carcinoma patients surviving more than 10 years after radical hepatectomy].

Objective: To clarify the clinicopathologic features of hepatocellular carcinoma (HCC) patients survived more than 10 years after radical hepatectomy. Methods: Two hundreds and fifty-two patients who underwent curative resection for HCC between January 1999 and March 2006 at Department of Hepatopancreatobiliary Surgery, Affiliated Hospital of Qingdao University were included.There were 217 male cases and 35 female cases aging from 17 to 82 years with median age of (53.8±10.5)years. Followed by March 31 2016, clinicopathologic factors in 10-year survivors and patients who died within 10 years were compared by χ(2) test, Kaplan-Meier survival analysis and Cox proportional hazards model and the prognostic factors affecting survival were identified. Results: All patients were followed-up for 4.0 to 205.7 months with median time of 53.4 months. The 10-year overall survival rate was 26%, there were 62 cases(26.2%) who survived for more than 10 years after initial hepatectomy. In survival >10-year group, the paitents with ALT<40 U/L, gamma-glutamyl transpeptidase<64 U/L, albumin≥35 g/L, without liver cirrhosis and portal hypertension, Child-Pugh grade A, no blood transfusion, AFP≤20 µg/L, tumor size ≤5.0 cm, single tumor, high differentiation, TNM stage Ⅰ and TACE negative after resection were more than the patients in survival <10-year group (P<0.05). In multivariate analysis, Child-Pugh grade A, the tumor size ≤5.0 cm and TACE negative after resection were favorable independent factors associated with 10-year survival (P<0.05). Conclusion: Based on the results of the study, Child-Pugh grade A, tumor size ≤5.0 cm and TACE negative after resection at initial hepatectomy might be biologically favorable conditions for patients surviving more than 10 years.

Impact of particulate matter 2.5 on the liver function of mice.

OBJECTIVE: The aim of this study was to evaluate the impact of particulate matter 2.5 (PM2.5) on liver function at the animal level and to study its impact targets. MATERIALS AND METHODS: 60 male and female BALB/c mice of SPF grade, aged 6-8 weeks, were randomly divided into four groups, with 15 mice in each, including the normal saline control group, the PM2.5 low dose group [2 µg/(100 g/d)], the PM2.5 medium dose group [8 µg/(100 g/d)] and the PM2.5 high dose group [16 µg/(100 g/d)]. Each day, 0.9% saline or PM2.5 particles were administered through the nasal route, and samples were taken after 3 weeks of continuous exposure. Hematoxylin-eosin staining (HE) was used to observe the liver damage caused by PM2.5. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by using an automatic biochemical analyzer to detect the content of liver glycogen and blood glucose. Multiple indicators were observed, including plasma tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) levels, oxidative stress response indicators reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) detection, RT-PCR and Western blot detection of glycogen synthase (GS), glucokinase (GK), nuclear factor erythroid 2-related factor 2 (Nrf2) expression and phosphorylation level of phospho-c-Jun N-terminal kinases (p-JNK). RESULTS: PM2.5 can cause damage to the liver by increasing PM2.5 concentrations, raising the metabolic rate of liver cells, resulting in a substantial amount of inflammatory infiltration and vacuolar degeneration of cells, and increasing the liver/body weight. TNF-α and IL-6 inflammatory factor expression increased (p<0.05). An increase in the serum ALT and AST levels were also observed. The blood glucose of mice increased, whereas the content of liver glycogen declined (p<0.05). ROS, MDA, and SOD levels all increased considerably. PM2.5 can drastically lower the expression of GS and GK, increase the expression of Nrf2, and raise the phosphorylation level of p-JNK (p<0.05). CONCLUSIONS: PM2.5 can induce oxidative stress in mouse liver through the Nrf2/JNK pathway, induce liver inflammation in mice, and inhibit glycogen synthesis.

[Effects of clinical application of free anterolateral thigh perforator lobulated flap in repair of electrical burn wounds on head based on the concept of donor site protection].

Objective: To explore the effects of clinical application of free anterolateral thigh perforator lobulated flap in repair of electrical burn wounds on head based on the concept of donor site protection. Methods: A retrospective observational study was conducted. Eight patients with electrical burns with huge scalp defects and exposed skulls were admitted to the First Affiliated Hospital of Zhengzhou University, from May 2017 to December 2019, who were all males, aged 21-57 (39±13) years, sustaining multiple deep partial thickness to full-thickness electrical burns to 5%-14% total body surface area. Among the scalp burn sites of the patients, 1 case was posterior occipital, 2 cases were parietal occipital, 4 cases were parietal temporal, and 1 case was frontotemporal. After debridement, the defect area was 10 cm×9 cm-16 cm×14 cm. The incision area of the free anterolateral thigh perforator lobulated flap was 22 cm×6 cm-30 cm×9 cm. The artery and vein of flap were anastomosed with superficial temporal artery and vein or facial artery and vein, and the other vein of skin flap was anastomosed with superficial vein of recipient area. The donor site of skin flap was closed by layer interrupted tension-reducing suture. After the operation, the survival of flop, donor site wound healing and complications were observed. The flap appearance, wound healing of donor sites, long-term complications and functional recovery of donor sites were observed on follow-up. Results: After the operation, the flaps of 8 patients survived completely without vascular crisis. The donor sites of flaps in all the patients healed well with no osteofascial compartment syndrome. Seven patients were followed up for 3 to 12 months, and 1 case was lost to follow up. During follow-up, the flaps of the patients' heads were in good appearance but with alopecia. The donor sites showed linear scars, which were well hidden. There were no significant differences in sensory and motor functions between the two sides, and no complications were found such as muscle hernia. Conclusions: Free anterolateral thigh perforator lobulated flap has a good clinical effect in the early repair of electrical burn wounds with huge scalp defect and skull exposure on head, and the donor wounds can be directly closed and sutured, greatly reducing the damage to the donor area.

[Clinical effects of concentrated growth factor combined with plasma albumin gel in treating facial depressed scar].

Objective: To explore the clinical effects of concentrated growth factor (CGF) combined with plasma albumin gel (PAG) in treating facial depressed scar. Methods: From January 2018 to June 2019, 14 patients in the First Affiliated Hospital of Zhengzhou University and 10 patients in Henan NO.3 Provincial People's Hospital with facial depressed scar who met the inclusion criteria were admitted, and their clinical data were retrospectively analyzed by the method of case-control study. Based on the method of treatment, 8 patients (4 males and 4 females) aged 28.50 (25.50, 31.50) years were enrolled in CGF alone group, 8 patients (3 males and 5 females) aged 32.00 (28.50, 35.00) years were enrolled in PAG alone group, and 8 patients (5 males and 3 females) aged 33.50 (29.00, 35.75) years were enrolled in CGF+ PAG group. Suitable amount of CGF, PAG, and CGF+ PAG (mixed at a ratio of 1.0∶1.0-1.0∶1.5) prepared from autologous blood were injected subcutaneously via a single or multiple entrance (s) into the depressed scar of patients in CGF alone, PAG alone, and CGF+ PAG groups respectively to fill up the concavity, once every 4 weeks for a total of 3 times. Before the first treatment (hereinafter referred to as before treatment) and 3 months after the last treatment (hereinafter referred to as after treatment), the Goodman & Baron Acne Scar Grading System was used for scar grading, and the difference was calculated; the Anxiety Self-Rating Scale was used to score anxiety, and the difference was calculated. The Visual Analogue Score was used to score pain immediately after the first treatment. By one, two, and three months after treatment, the patients' satisfaction to scar treatment was scored, and the Global Aesthetic Improvement Scale was used to score the scar improvement. Adverse reaction of patients after treatment was monitored. Data were statistically analyzed with Fisher's exact probability test, Kruskal-Wallis H test, Mann-Whitney U test, Bonferroni correction, and Wilcoxon signed rank sum test. Results: (1) The scars of patients in the three groups were all graded 4.00 (4.00, 4.00) before treatment (χ(2)<0.001, P>0.05). By three months after treatment, compared with 2.00 (1.25, 2.00) of CGF alone group, the scar grades of patients in PAG alone group and CGF+ PAG group (3.00 (2.00, 3.00) and 1.00 (1.00, 1.00), respectively) had no significant change (Z=2.199, 2.003, P>0.05). The scar grade of patients in CGF+ PAG group was significantly lower than that in PAG alone group (Z=3.229, P<0.01). Compared with those before treatment, the scar grades of patients in CGF alone group, PAG alone group, and CGF+ PAG group were significantly reduced three months after treatment (Z=2.588, 2.598, 2.640, P<0.05 or P<0.01). The difference in scar grade before and after the treatment was significantly higher in CGF+ PAG group than in PAG alone group (Z=3.229, P<0.01). (2) The anxiety scores of patients in the three groups were similar before treatment and 3 months after (χ(2)=2.551, 2.768, P>0.05). Compared with those before treatment, the anxiety scores of patients in CGF alone group, PAG alone group, and CGF+ PAG group were significantly reduced three months after treatment (Z=2.395, 2.527, 2.533, P<0.05). The differences in anxiety score before and after the treatment were similar among the three groups (χ(2)=1.796, P>0.05). (3) The pain scores of patients in the three groups were similar immediately after the first treatment (χ(2)=0.400, P>0.05). (4) By one and two month (s) after treatment, the patients' satisfaction scores to scar treatment in the three groups were similar (χ(2)=2.688, 5.989, P>0.05). By three months after treatment, the patients' satisfaction score to scar treatment in CGF+ PAG group was significantly higher than that in PAG alone group (Z=2.922, P<0.01). Compared with those one month after treatment within the same group, the patients' satisfaction scores to scar treatment in CGF alone group, PAG alone group, and CGF+ PAG group were significantly increased two and three months after treatment (Z=1.121, 2.392, 2.000, 2.828, 2.449, 2.598, P<0.05 or P<0.01). Compared with those two months after treatment within the same group, the patients' satisfaction scores to scar treatment in CGF alone group, PAG alone group, and CGF+ PAG group were significantly increased three months after treatment (Z=2.271, 2.000, 2.646, P<0.05 or P<0.01). (5) One month after treatment, the scar improvement scores of patients in the three groups were similar (χ(2)=4.438, P>0.05). Two months after treatment, the scar improvement scores of patients in CGF alone group and CGF+ PAG group were 2.00 (2.00, 2.75) and 2.00 (2.00, 2.00) points, respectively, which were significantly higher than 1.00 (1.00, 1.00) point of PAG alone group (Z=3.303, 3.771, P<0.01). Three months after treatment, the scar improvement score of patients in CGF+ PAG group was 3.00 (3.00, 3.00) points, which was significantly higher than 2.00 (2.00, 2.75) points of CGF alone group and 1.00 (1.00, 2.00) points of PAG alone group (Z=2.450, 3.427, P<0.05 or P<0.01). Compared with those one month after treatment within the same group, the scar improvement scores of patients were significantly higher in CGF alone group and CGF+ PAG group two and three months after treatment and in PAG alone group three months after treatment (Z=2.828, 2.828, 2.530, 2.640, 2.121, P<0.05 or P<0.01). Compared with that two months after treatment within the same group, the scar improvement score of patients in CGF+ PAG group was significantly higher three months after treatment (Z=2.449, P<0.05). (6) After injection, all patients in the three groups had slight redness and swelling at the needle prick point and no other adverse reactions. Conclusions: CGF combined with PAG can reduce the scar grading, anxiety of patients, and enhance patients' satisfaction and scar improvement in the treatment of patients with facial depressed scar. The combined CGF+ PAG injection, without significant adverse reactions, is better than single component injection and is worthy of clinical application.

C-MYC-induced upregulation of LINC01503 promotes progression of non-small cell lung cancer.

OBJECTIVE: The purpose of this study was to detect the expression of long intergenic non-protein-coding RNA 1503 (LINC01503) in non-small cell lung cancer (NSCLC), and to further study its biological function, as well as the regulatory relationships of c-MYC with LINC01503 and the extracellular signal regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway in NSCLC. PATIENTS AND METHODS: Tissue specimens were collected from 36 NSCLC patients, and the relative expression level of LINC01503 in the 36 cases of NSCLC tissue specimens and NSCLC cells was then determined using quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Then, the effects of LINC01503 on the proliferation and apoptosis of NSCLC cells were detected in vitro via Cell-Counting Kit (CCK)-8 assay, colony-forming assay and flow cytometry. Besides, the possible LINC01503 promoter-binding transcription factor was predicted using bioinformatics. After interference with c-MYC expression, the changes in the expression of LINC01503 were examined through qRT-PCR. Finally, the changes in the expressions of the molecular markers in the ERK/MAPK signaling pathway after interference with LINC01503 and c-MYC expressions were evaluated using Western blotting. RESULTS: According to qRT-PCR results, the expression of LINC01503 was upregulated in 30 out of 36 cases of NSCLC tissues. Compared with that in human normal bronchial epithelial cells, the expression of LINC01503 was elevated in NSCLC cells. As shown by the CCK-8 assay and colony-forming assay, the proliferation ability of NSCLC cells was weakened after interference with LINC01503 expression, and the flow cytometry results revealed the apoptosis rate of NSCLC cells was raised after interference with LINC01503 expression. Moreover, the bioinformatics prediction showed that c-MYC might be the LINC01503 promoter-binding transcription factor. Additionally, it was found through the qRT-PCR that the expression of LINC01503 declined after interference with c-MYC expression. Finally, based on Western blotting results, the expressions of phosphorylated ERK1/2 (p-ERK1/2) and p-MAPK/ERK kinase (MEK), the molecular markers in the ERK/MAPK signaling pathway, were inhibited after interference with c-MYC and LINC01503 expressions. CONCLUSIONS: The transcription factor c-MYC promotes the expression of LINC01503 in NSCLC and activates the ERK/MAPK signaling pathway to drive the development and progression of NSCLC.

[Clinical effects of superior gluteal artery perforator " buddy flap" in repairing pressure ulcer in sacrococcygeal region].

Objective: To investigate the clinical effects of superior gluteal artery perforator " buddy flap" in repairing pressure ulcer in sacrococcygeal region. Methods: From January 2017 to December 2018, 13 patients (8 males and 5 females) aged 24-79 years with stage 4 pressure ulcers in sacrococcygeal region were admitted to the First Affiliated Hospital of Zhengzhou University, with wound area from 5 cm×4 cm to 12 cm×10 cm. After thorough debridement and vacuum sealing drainage, the superior gluteal artery perforator " buddy flap" was designed to repair the pressure ulcer in sacrococcygeal region. The pressure ulcer was repaired by the main flap with area from 7.0 cm×5.0 cm to 18.0 cm×12.0 cm; the main flap's donor area was covered by the auxiliary flap with area from 5.0 cm×3.0 cm to 11.0 cm×7.0 cm; the auxiliary flap's donor area was covered by the connecting flap between the main flap and the auxiliary flap. The remaining wound without covering was directly closed by suturing. The postoperative flap survival and complications were observed. The appearance and function of flaps and the recurrence of pressure ulcer were followed up. Results: The flaps of 12 patients survived after operation without complications of infection, fat liquefaction, or poor flap survival. A small area of superficial necrotic skin at the distal end of flap was observed in one case, which was healed after dressing change. All the patients were followed up for 6 months without recurrence of pressure ulcer, and the operation area was naturally full in appearance, which was pressure and wear resistant. Conclusions: Superior gluteal artery perforator " buddy flap" is an effective method for the treatment of pressure ulcer in sacrococcygeal region. The effect of tension-free repair of the pressure ulcer and main flap donor area can be achieved in one operation. The operation is simple, the curative effect is accurate, and it has certain clinical value.

[Meta-analysis of the effects of triamcinolone acetonide alone and in combination with 5-fluorouracil for treating keloids].

Objective: To compare the efficacy and safety of triamcinolone acetonide (TA) alone and in combination with 5-fluorouracil (5-FU) for treating keloids using meta-analysis. Methods: Databases including PubMed, Embase, and Cochrane Library were retrieved with the search terms of " triamcinolone acetonide, 5-fluorouracil, glucocorticoid, fluorouracil, keloid, scar, TAC, 5-FU, hypertrophic scar " and databases including Chinese Journal Full-Text Database, Chinese Biomedical Database, and Wanfang Data were retrieved with the search terms of ",, 5-,," in Chinese to obtain the publicly published randomized controlled trials about the effects of TA alone and in combination with 5-fluorouracil for treating keloids from the establishment of each database to august 2019. The outcome indexes included effective proportion of treatment, incidence proportion of adverse reactions, and recurrence proportion of keloids. RevMan 5.3 and Stata 14.0 statistical software were used to conduct a meta-analysis of eligible studies. Results: A total of 1 326 patients with keloids were included in 14 studies, including 668 patients in TA+ 5-fluorouracil group whose keloids were injected with TA and 5-fluorouracil and 658 patients in TA alone group whose keloids were injected with TA alone. A total of 7 articles achieved 1 to 3 points in modified Jadad score, while 7 articles achieved 4 to 7 points in modified Jadad score. Patients in TA+ 5-fluorouracil group had a higher effective proportion of treatment than that of TA alone group (relative risk=1.28, 95% confidence interval=1.16-1.41, P<0.01). Subgroup analysis showed that the quality of the included literature and ethnic factors might be the source of heterogeneity in effective proportion of treatment. Patients in TA+ 5-fluorouracil group had a lower incidence proportion of adverse reactions than that of TA alone group (relative risk=0.44, 95% confidence interval=0.25-0.75, P<0.01). Patients in TA+ 5-fluorouracil group had a lower recurrence proportion of keloids than that of TA alone group (relative risk=0.25, 95% confidence interval=0.14-0.44, P<0.01). There was no publication bias in incidence proportion of adverse reactions (P>0.05), while the effective proportion of treatment and recurrence proportion of keloids had publication bias (P<0.05). Conclusions: TA combined with 5-fluorouracil is more effective than TA alone for treating keloids, with less incidence of adverse reactions and recurrence.

Downregulated miR-328 suppressed cell invasion and growth in hepatocellular carcinoma via targeting PTEN.

OBJECTIVE: To investigate the regulatory effect of miR-328 on biological behaviors of hepatocellular carcinoma (HCC) cells, such as invasion and proliferation. PATIENTS AND METHODS: The expressions of miR-328 were detected in 48 pairs of HCC tissue samples and matched adjacent tissues, as well as in 3 kinds of HCC cell lines via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Further, we analyzed the effects of miR-328 inhibition on cell invasion, proliferation, cell apoptosis, and cell cycle. Dual-luciferase activity assay was performed to examine the potential target gene PTEN which was predicted by an online database. Protein levels were detected using Western blot assay. RESULTS: The expression of miR-328 was significantly increased in HCC tissue samples. Decreased miR-328 in HCC cells significantly attenuated cell invasion and proliferation capacities, promoted cell apoptosis and induced cell cycle arrest at G0/G1 phase. Moreover, PTEN was verified as a target gene of miR-328 by dual-luciferase activity assay, qRT-PCR and Western blot. Furthermore, the silence of PTEN neutralized the suppressive effect of decreased miR-328 on cell growth and metastasis. CONCLUSIONS: MiR-328 is involved in the development of HCC via regulating PTEN, which might provide a new target for HCC diagnosis and therapy.

[Advances in the research of effects of glutamine on immune function of burn patients].

Glutamine is the most abundant amino acid found in plasma and cells. It is the preferred fuel for enterocytes in the small intestine, macrophages, and lymphocytes. After serious burn, increased requirement of glutamine by the gastrointestinal tract, kidney and lymphocytes, and relatively insufficient self synthesis likely contribute to the rapid decline of glutamine in circulation and cells. Glutamine supplementation can not only protect intestinal mucosa, maintain normal intestinal barrier function, reduce bacterial translocation, and enhance the intestinal immune function, but also increase the number of lymphocytes, enhance the phagocytic function of macrophage, promote the synthesis of immunoglobulin, and reduce the body's inflammatory response, so as to enhance the immune function. Therefore, glutamine supplementation can improve and enhance the immune function, reduce complications and promote the prognosis of severely burned patients.

[Application of percutaneous transluminal angioplasty in the surgical treatment of patients with diabetic feet].

OBJECTIVE: To explore the application of percutaneous transluminal angioplasty (PTA) in the surgical treatment of patients with diabetic feet. METHODS: The clinical data of 83 patients with diabetic feet, 95 limbs (95 wounds) in total, hospitalized in our unit from September 2011 to September 2014, conforming to the study criteria, were retrospectively analyzed. Patients were divided into conventional treatment group (CT, n=43, 51 wounds) and PTA group (n=40, 44 wounds) according to whether receiving PTA treatment or not. Patients in two groups received conventional debridement after admission, and patients in PTA group received another PTA treatment before debridement. Granulation growing well rates of wounds of patients in two groups were calculated on post debridement day (PDD) 3, 6, 9, and 12. Two stage preoperative preparation time of wounds of patients in two groups was recorded. Status of free skin graft survival of wounds and wound healing of patients in two groups were recorded according to the grade of Wagner. Values of ankle-brachial index (ABI) and ulcer recurrence of patients in two groups checked every month during follow-up time of half a year were recorded. Data were processed with chi-square test and t test. RESULTS: Granulation growing well rate of wounds of patients in group CT rose slowly after treatment, which was less than 40% on PDD 12. Granulation growing well rate of wounds of patients in PTA group rose significantly on PDD 9 and all the granulation grew well on PDD 12. On PDD 9 and 12, Granulation growing well rates of wounds of patients in PTA group were significantly higher than those in group CT (with χ(2) values respectively 30.008 and 47.810, P values below 0.01). Two stage preoperative preparation time of wounds of patients in group CT [(24±10) d] was obviously longer than that in PTA group [(15±3) d, t=5.709, P<0.01]. The ratios of survived free skin graft and healed suture in Wagner 2, 3, and 4 wounds of patients in PTA group were significantly higher than those in corresponding Wagner of group CT (with χ(2) values from 6.741 to 24.498, P values below 0.01). During follow-up time of half a year, values of ABI of patients in PTA group every month were significantly higher than those in group CT (with t values from 5.411 to 9.583, P values below 0.01). During follow-up time of half a year, there was no ulcer recurrence in group CT in the first four months, but ulcer recurrence was observed in one patient in the fifth month and in two patients in the sixth month. While no ulcer recurrence was found in PTA group during follow-up time of half a year. CONCLUSIONS: PTA has certain effect and clinical value for the treatment of diabetic foot.

KN-62, a calcium/calmodulin-dependent protein kinase II inhibitor, inhibits high potassium-stimulated prolactin secretion and intracellular calcium increases in anterior pituitary cells.

In isolated rat anterior pituitary cells, KN-62 (10 microM), an isoquinoline sulfonamide inhibitor of calcium/calmodulin-dependent protein kinase II, inhibited high KCl(50 milliM)-stimulated prolactin secretion almost completely, with an IC50 of 95 nM KN-62 inhibited TRH-induced prolactin secretion less effectively. KN-04, a compound that is over 100-fold less active in inhibiting purified calcium/calmodulin-dependent protein kinase II, also inhibited high KCl-stimulated prolactin secretion with an IC50 of 500 nM. KN-62 and KN-04 (10 microM) both inhibited high KCl-stimulated increases in intracellular Ca2+ concentrations. We conclude that KN-62 and KN-04 inhibit activation of voltage-dependent calcium channels in anterior pituitary cells either directly or indirectly.

Pyrrolidine dithiocarbamate restores gastric damages and suppressive autophagy induced by hydrogen peroxide.

It is well known that gastric barrier is very important for protecting host from various insults. Simultaneously, autophagy serving as a prominent cytoprotective and survival pathway under oxidative stress conditions is being increasingly recognized. Thus, this study was conducted for investigating the effect of pyrrolidine dithiocarbamate (PDTC) on gastric barrier function and autophagy under oxidative stress induced by intragastric administration of hydrogen peroxide (H2O2). The gastric tight junction proteins [zonula occludens-1 (ZO1), occludin, and claudin1], autophagic proteins [microtubule-associated protein light chain 3I(LC3I), LC3II, and beclin1], and nuclear factor kappa B (NF-κB) signaling pathway (p65 and IκB kinase α/ß) were determined by Western blot. The results showed that H2O2 exposure disturbed gastric barrier function with decreased expression of ZO1, occludin, and claudin1, and reduced gastric autophagy with decreased conversion of LC3I into LC3II in mice. However, treatment with PDTC restored these adverse effects evidenced by increased expression of ZO1 and claudin1 and increased conversion of LC3I into LC3II. Meanwhile, H2O2 exposure decreased normal human gastric epithelial mucosa cell line (GES-1) viability in a concentration-dependent way. However, after being exposed to H2O2, GES-1 exhibited autophagic response which was inconsistent with our in vivo results in mice, while PDTC failed to decrease autophagy in GES-1 induced by H2O2. Simultaneously, the beneficial effect of PDTC on gastric damage and autophagy in mice might be independent of inhibition of NF-κB. In conclusion, PDTC treatment restores gastric damages and reduced autophagy induced by H2O2. Therefore, PDTC may serve as a potential adjuvant therapy for gastric damages.

Calcium/calmodulin-dependent protein kinase-II activation in rat pituitary cells in the presence of thyrotropin-releasing hormone and dopamine.

PRL release from rat lactotrophs in response to TRH is Ca2+ dependent. TRH-induced PRL release is inhibited either after repeated pulses of TRH or in the presence of dopamine. TRH, however, generates increases in intracellular Ca2+ concentrations ([Ca2+]i) in both conditions. Calcium/calmodulin-dependent protein kinase-II (CaM kinase-II) is a ubiquitous enzyme implicated in secretion. To determine whether down-regulation of CaM kinase-II activity caused the lack of responsiveness to increases in [Ca2+]i, we measured the generation of calcium/calmodulin-independent kinase activity. Anterior pituitary cells contain a 50-kilodalton form of CaM kinase-II, determined by immunoblot, and the enzyme is in lactotrophs, determined by immunocytochemistry. TRH rapidly and transiently increased calcium/calmodulin-independent kinase activity; the increase was maximal by 15 sec and returned to basal by 2 min. When TRH pulses (1 microM, 15 sec) were applied every 10 min, each pulse caused an increase in calcium/calmodulin-independent kinase activity of similar magnitude, and the activity returned to basal values between pulses. Pretreatment of cells with dopamine (1 microM; 30 min) inhibited PRL release, but did not prevent the increase in calcium/calmodulin-independent kinase activity. These results indicate that TRH still activates CaM kinase-II when PRL release is inhibited. Dopamine and repeated pulses of TRH must inhibit PRL release at a site after the TRH-induced increase in [Ca2+]i and at a site other than CaM kinase-II.

Photodynamic action of rose bengal on isolated rat pancreatic acini: stimulation of amylase release.

The halogenated fluorescein derivative, rose bengal, upon photon activation, elicits amylase secretion from isolated, perifused pancreatic acini. This effect is due to production of highly reactive singlet delta oxygen which can permeabilize the cell membrane and may also react chemically with secretagogue receptors, or other functional components of the membrane such as the G-proteins. The profile of photodynamically induced amylase secretion is anion-dependent: it becomes biphasic when the chloride ion is substituted by the glutamate ion, an effect attributed to the action of glutamate on the ionic transport systems of the zymogen granule membrane.

Types of voltage-dependent calcium channels involved in high potassium depolarization-induced amylase secretion in the exocrine pancreatic tumour cell line AR4-2J.

In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted large amount of amylase. High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation. High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel antagonists with an order of potency as follows: nifedipine > omega-agatoxin IVA > omega-conotoxin GVIA. In contrast, the L-type calcium channel antagonist nifedipine almost completely inhibited potassium-induced amylase secretion, whereas the N-type channel antagonist omega-conotoxin GVIA was without effect. The P-type channel antagonist omega-agatoxin IVA had a small inhibitory effect, but this inhibition was not significant at the level of amylase secretion. In conclusion, the AR4-2J cell line possesses different voltage-dependent calcium channels (L, P, N) with the L-type predominantly involved in depolarization induced amylase secretion.

Photodynamic action of sulphonated aluminium phthalocyanine (SALPC) on isolated rat pancreatic acini.

The photodynamic action of SALPC has been investigated on dispersed, perifused, acini isolated from the rat pancreas. Stimulation of secretion was assessed by measuring amylase release and membrane permeabilization determined by the leakage of cytoplasmic lactate dehydrogenase (LDH) and by the efflux of 86Rb from preloaded acini. Light alone (greater than 570 nm, less than or equal to 18,400 lux), or SALPC (less than or equal to 1 microM) in the absence of light, had no effect on pancreatic acini but cellularly bound SALPC when illuminated caused a dose-dependent, light intensity-dependent and temperature-dependent release of amylase. Singlet oxygen generated by photon-activation of SALPC was measured by the formation of an imidazole adduct and bleaching of the secondary substrate, RNO. Whereas illumination caused a rapid increase in photodynamically-evoked pancreatic amylase release, the efflux of 86Rb and loss of cytosolic LDH were markedly delayed in onset: similar results were obtained with monochromatic laser light (633 nm). In contrast, the muscarinic agonist bethanechol evoked a rapid increase in amylase release but with an almost immediate efflux of 86Rb. Finally, electron microscopy confirmed that the structural integrity of the pancreatic acinar cells was maintained after the photodynamic action of SALPC. It is concluded that the stimulation of amylase secretion and membrane permeabilization by SALPC is due to the generation of singlet oxygen. However, the consistent difference between the time course of amylase secretion and membrane permeabilization makes it likely that an initial stage in photodynamic drug action involves oxidation of plasma membrane protein and activation of secretagogue receptors or the G-proteins and their effector systems.

Photodynamic drug action on isolated rat pancreatic acini. Mobilization of arachidonic acid and prostaglandin production.

Chloro-aluminium phthalocyanine sulphonate (SALPC) when photon-activated generates singlet oxygen, elicits amylase release and causes plasma membrane permeabilization of pancreatic acinar cells (Matthews and Cui, Biochem Pharmacol 39: 1444-1457, 1990). Amylase release precedes membrane permeabilization suggesting that the initial release of amylase may be due to direct stimulation by singlet oxygen of secretagogue receptors or their coupled guanine nucleotide binding proteins (G-proteins) and effector systems including phospholipase A2 (PLA2). The aim of the experiments reported here was to establish the extent to which PLA2 activation, arachidonic acid mobilization, and prostaglandin production are involved in the photon-induced action of SALPC on dispersed, perifused acini isolated from the rat pancreas. The mobilization of arachidonic acid by a major secretory stimulant of pancreatic exocrine cells, cholecystokinin octapeptide, was also assessed: it produced a time- and concentration-dependent (10(-10)-10(-6) M) stimulation of arachidonic acid output from acini prelabelled with [1-14C]arachidonic acid. In contrast, the kinetics of arachidonic acid mobilization with photon-activated SALPC 1 microM, 4500 or 18,400 lux light intensity (lambda > 570 mm), was biphasic, an intensity-dependent stimulation being preceded by a more immediate initial inhibition of output. Light activation of SALPC and singlet oxygen generation may evoke the stimulatory phase of arachidonic acid release by an action on G-proteins, or by PLA2 activated directly, or via calcium influx, because NaF 20 mM, mellitin 2 mg/mL and the calcium ionophore A23187 1 microM caused a 2.9-, 33- and 5-fold increase, respectively, in arachidonic acid output. However, not only was the arachidonate stimulation delayed in response to SALPC but in other experiments designed to gain more insight into the turnover of arachidonic acid and its metabolites, the photodynamic release of amylase preceded maximum prostaglandin E2 (PGE2) output and amylase release was completely unaffected when PGE2 production was blocked by the cyclo-oxygenase inhibitor, indomethacin 10 microM. It is therefore likely that the rapid initial photodynamic release of amylase from pancreatic acini induced by SALPC is mediated by activation of the signal transduction pathway involving the release of intracellular calcium; arachidonic acid mobilization and prostanoid production may then be linked to the longer-term, cytolytic action of SALPC, especially in tumour cells.

A novel aspect of photodynamic action: induction of recurrent spikes in cytosolic calcium concentration.

Effects of photodynamic action of gadolinium porphyrin-like macrocycle B (PLMGdB) on cytosolic Ca2+ concentration, [Ca2+]c, was investigated in isolated rat pancreatic acini. The PLMGdB alone or light alone (2 min) had no effect on [Ca2+]c. Cell-bound PLMGdB upon brief (0.5-2.0 min) light activation triggered recurrent spikes in [Ca2+]c. At lower PLMGdB concentration (100 nM) the spikes continued during the whole period of monitoring [Ca2+]c. At a higher concentration of 500 nM, the spikes continued for the first 40 min, followed by a gradual increase in basal [Ca2+]c upon which smaller spikes were superimposed. At 1 microM, the spikes continued for the first 20 min, after that spiking gradually degenerated into a plateau phase. In many aspects, photodynamically triggered spikes resembled spikes generated by physiological concentrations of cholecystokinin. The spikes triggered by photodynamic action were likely to be the result of the ignition of a physiological "chain reaction", because functional inositol-1,4,5-trisphosphate (IP3) receptors were required for spiking to occur. Two-aminoethoxydiphenylborate, an inhibitory modulator of IP3-triggered Ca2+ release from intracellular stores, effectively inhibited photodynamically generated spikes. Therefore photodynamic action appears to be able to permanently transfix a physiological process, leading to long-lasting pharmacological or therapeutic effects.

Photodynamic action inhibits compound 48/80-induced exocytosis in rat peritoneal mast cells.

Photostimulation of sulfonated aluminum phthalocyanine (SALPC)-loaded mast cells (20,000 lux, 2 min) itself caused neither exocytosis nor [Ca2+]i increase in isolated rat peritoneal mast cells. This result is incompatible with that reported in other cell types such as pancreatic acinar cells. Stimulation with 50 micrograms/ml compound 48/80, a direct G-protein activator, induced massive exocytosis which was easily detectable under conventional microscope. The fluorescent granules stained with sulforhodamine B were found to be numerous on the perimetry of mast cells, confirming occurrence of exocytosis. The stimulation also increased [Ca2+]i and cell volume before initiation of exocytosis. Pretreatment of the cells with photodynamic action with 5 microM SALPC inhibited the compound 48/80-induced exocytosis, but the [Ca2+]i increase and the increase of cell volume were unaffected. NaN3 at 0.5 mM could relieve the photodynamic action-induced inhibition of exocytosis. These results indicate that, unlikely to other secretory or contractile cells, photodynamic action with SALPC does not directly affect exocytotic machinery but modulates some functional proteins involved in signal transduction process which may be posterior to G-protein activation in mast cells. Singlet oxygen may be involved in the photodynamic action-induced modulation. A possible target protein can be a protein in the cell membrane which binds with a protein of a granular membrane during the course of exocytosis.