Functionally Collaborative Nanostructure for Direct Monitoring of Neurotransmitter Exocytosis in Living Cells.

Neurotransmitter exocytosis of living cells plays a vital role in neuroscience. However, the available amperometric technique with carbon fiber electrodes typically measures exocytotic events from one cell during one procedure, which requires professional operations and takes time to produce statistical results of multiple cells. Here, we develop a functionally collaborative nanostructure to directly measure the neurotransmitter dopamine (DA) exocytosis from living rat pheochromocytoma (PC12) cells. The functionally collaborative nanostructure is constructed of metal-organic framework (MOF)-on-nanowires-on-graphene oxide, which is highly sensitive to DA molecules and enables direct detection of neurotransmitter exocytosis. Using the microsensor, the exocytosis from PC12 cells pretreated with the desired drugs (e.g., anticoronavirus drug, antiflu drug, or anti-inflammatory drug) has been successfully measured. Our achievements demonstrate the feasibility of the functionally collaborative nanostructure in the real-time detection of exocytosis and the potential applicability in the highly efficient assessment of the modulation effects of medications on exocytosis.

Protective effects of safranal on diabetic retinopathy in human microvascular endothelial cells and related pathways analyzed with transcriptome sequencing.

Aim: To determine the effect of safranal on diabetic retinopathy in vitro and its possible mechanisms. Methods: We used human retinal microvascular endothelial cells (HRMECs) to test the influence of safranal in vitro. High glucose damage was established and an safranal was tested at various concentrations for its potential to reduce cell viability using the MTT assay. We also employed apoptosis detection, cell cycle detection, a transwell test, and a tube formation assay to look into safranal's inhibitory effects on high glucose damage at various doses. Furthermore, mRNA transcriptome sequencing was performed. mRNA expression levels in a high glucose damage model, a high glucose damage model treated with safranal, and a blank control were compared to find the possible signaling pathway. Western blotting was used to confirm the expressions of several molecules and the levels of phosphorylation in each for the newly discovered pathway. Results: Cell proliferation was inhibited under a high glucose condition but could be protected by safranal at different concentrations (P<0.001). Flow cytometry results suggested safranal also protected cells from apoptosis (P=0.006). A transwell test demonstrated reduced invasiveness of safranal-treated cells in a high glucose condition (P<0.001). In a tube formation investigation, there were noticeably more new branches in the high gloucose group compared to a high glucose treated with safranal group (P<0.001). In mRNA expression patterns on transcriptome sequencing, the MAPK signaling pathway showed an expression ratio. With western blotting, the phosphorylation level of p38-AKT was elevated under a high glucose condition but could be inhibited by safranal. The expression of molecules associated with cell adhesion, including E-cadherin, N-cadherin, Snail, Twist, and fibronectin also changed significantly after safranal treatment under a high glucose condition. Conclusion: Safranal can protect diabetic retinopathy in vitro, and the p38-AKT signaling pathway was found to be involved in the pathogenesis of diabetic retinopathy and could be inhibited by safranal. This pathway may play a role by influencing cell migration and adhesion.

KMT2D deficiency enhances the anti-cancer activity of L48H37 in pancreatic ductal adenocarcinoma.

BACKGROUND: Novel therapeutic strategies are urgently needed for patients with a delayed diagnosis of pancreatic ductal adenocarcinoma (PDAC) in order to improve their chances of survival. Recent studies have shown potent anti-neoplastic effects of curcumin and its analogues. In addition, the role of histone methyltransferases on cancer therapeutics has also been elucidated. However, the relationship between these two factors in the treatment of pancreatic cancer remains unknown. Our working hypothesis was that L48H37, a novel curcumin analog, has better efficacy in pancreatic cancer cell growth inhibition in the absence of histone-lysine N-methyltransferase 2D (KMT2D). AIM: To determine the anti-cancer effects of L48H37 in PDAC, and the role of KMT2D on its therapeutic efficacy. METHODS: The viability and proliferation of primary (PANC-1 and MIA PaCa-2) and metastatic (SW1990 and ASPC-1) PDAC cell lines treated with L48H37 was determined by CCK8 and colony formation assay. Apoptosis, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, and cell cycle profile were determined by staining the cells with Annexin-V/7-AAD, JC-1, DCFH-DA, and PI respectively, as well as flow cytometric acquisition. In vitro migration was assessed by the wound healing assay. The protein and mRNA levels of relevant factors were analyzed using Western blotting, immunofluorescence and real time-quantitative PCR. The in situ expression of KMT2D in both human PDAC and paired adjacent normal tissues was determined by immunohistochemistry. In vivo tumor xenografts were established by injecting nude mice with PDAC cells. Bioinformatics analyses were also conducted using gene expression databases and TCGA. RESULTS: L48H37 inhibited the proliferation and induced apoptosis in SW1990 and ASPC-1 cells in a dose- and time-dependent manner, while also reducing MMP, increasing ROS levels, arresting cell cycle at the G2/M stages and activating the endoplasmic reticulum (ER) stress-associated protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 (ATF4)/CHOP signaling pathway. Knocking down ATF4 significantly upregulated KMT2D in PDAC cells, and also decreased L48H37-induced apoptosis. Furthermore, silencing KMT2D in L48H37-treated cells significantly augmented apoptosis and the ER stress pathway, indicating that KMT2D depletion is essential for the anti-neoplastic effects of L48H37. Administering L48H37 to mice bearing tumors derived from control or KMT2D-knockdown PDAC cells significantly decreased the tumor burden. We also identified several differentially expressed genes in PDAC cell lines expressing very low levels of KMT2D that were functionally categorized into the extrinsic apoptotic signaling pathway. The KMT2D high- and low-expressing PDAC patients from the TCGA database showed similar survival rates,but higher KMT2D expression was associated with poor tumor grade in clinical and pathological analyses. CONCLUSION: L48H37 exerts a potent anti-cancer effect in PDAC, which is augmented by KMT2D deficiency.

Identification and functional analysis of an iron-binding protein, ferritin heavy chain subunit, from the swallowtail butterfly, Papilio xuthus.

Ferritin, which is ubiquitous among all living organisms, plays a crucial role in maintaining iron homeostasis, immune response, and detoxification. In the present research, we identified an iron-binding protein, ferritin heavy chain subunit, from Papilio xuthus and named PxFerHCH. The complete complementary DNA of PxFerHCH was 1,252 bp encoding a sequence of 211 amino acids, which includes an iron-responsive element. Phylogenetic analysis showed that PxFerHCH is clustered with Manduca sexta and Galleria mellonella ferritin heavy chain subunits. Expression levels of PxFerHCH in various tissues were analyzed by reverse transcription quantitative polymerase chain reaction, and the results exhibited that PxFerHCH was expressed in all tissues with the highest expression in the fat body. The relative expression level of PxFerHCH in response to bacterial (Escherichia coli and Staphylococcus aureus) challenges sharply increased by about 12 hr postinfection (hpi) and then decreased at 24 hpi. In addition, the iron-binding capacity and antioxidation activity of recombinant PxFerHCH protein were also investigated. These results reveal that PxFerHCH might play an important role in defense against bacterial infection.

GRGM-13 comprising 13 plant and animal products, inhibited oxidative stress induced apoptosis in retinal ganglion cells by inhibiting P2RX7/p38 MAPK signaling pathway.

OBJECTIVE: To determine the effectiveness of GRGM-13 on oxidative stress induced apoptosis of retinal ganglion cells (RGCs) and revealed its possible mechanism. MATERIALS AND METHODS: Caspase-3 activity, MDA level, and glutathione peroxidase level were detected by Caspase-3 assay kit, Lipid Peroxidation MDA Assay Kit, and Total Glutathione Peroxidase Assay Kit, respectively. Protein levels of Bax, Bcl-2, p-p38 and p38 were observed by Western Blot. Reactive oxygen species assay kit was used to determine intracellular ROS level. Apoptotic cells were measured by flow cytometry. RESULTS: GRGM-13 inhibited apoptosis of RGCs and ROS level in rat retinal tissue and RGC-5 cells, and the decrease degree strengthened with the increase of GRGM-13 concentration. In addition, ROS upregulated p-p38 expression, while GRGM-13 reversed this effect. We also found that p38 inhibitor SB202190 did not change L-glutamate (Glu) or H2O2-induced ROS level, while SB202190 inhibited apoptosis of RGC-5 cells. Finally, we observed that P2 × 7R agonist BzATP reversed the inhibition effect of GRGM-13 on RGC-5 cell apoptosis, ROS level and p-p38 expression, while si-P2 × 7R inhibited oxidative stress-induced phosphorylation of p38. CONCLUSION: GRGM-13 could inhibit oxidative stress-induced RGCs apoptosis via inhibiting P2RX7/p38 MAPK pathway, which revealed the possible mechanism of GRGM-13 on stress-induced RGCs apoptosis and provided new Chinese medicine for the treatment of glaucoma.

MicroRNA-155 promotes the pathogenesis of experimental colitis by repressing SHIP-1 expression.

AIM: To explore the mechanism by which microRNA-155 (miR-155) regulates the pathogenesis of experimental colitis. METHODS: A luciferase assay was performed to confirm the binding of miR-155 to the SHIP-1 3'-UTR. MiR-155 mimics, negative controls and SHIP-1 expression/knockdown vectors were established and then utilized in gain- and loss-of-function studies performed in raw264.7 cells and primary bone marrow-derived macrophages (BMDMs). Thereafter, dextran sulfate sodium (DSS)-induced colitis mouse model with or without antagomiR-155 treatment was established, and the levels of miR-155 and SHIP-1, as well as the pro-inflammatory capabilities, were measured by western blot, quantitative polymerase chain reaction, and immunohistochemistry. RESULTS: MiR-155 directly bound to the 3'-UTR of SHIP-1 mRNA and induced a significant decrease in SHIP-1 expression in both raw264.7 cells and primary BMDMs. MiR-155 markedly promoted cell proliferation and pro-inflammatory secretions including IL-6, TNF-α, IL-1ß, and IFN-γ, whereas these effects could be reversed by the restoration of SHIP-1 expression. In vivo studies showed that antagomiR-155 administration could alleviate DSS-induced intestinal inflammation in Balb/c mice. Moreover, significantly increased SHIP-1 expression, as well as decreased Akt activation and inflammatory response, were observed in the antagomiR-155-treated mice. CONCLUSION: MiR-155 promotes experimental colitis by repressing SHIP-1 expression. Thus, the inhibition of miR-155 might be a promising strategy for therapy.

Down-regulation of microRNA-155 attenuates retinal neovascularization via the PI3K/Akt pathway.

PURPOSE: We aimed to investigate the anti-angiogenic properties of miR-155 via in vitro and in vivo studies. METHODS: miR-155 was knocked down using lentivirus-mediated RNA interference. The proliferation, migration, and tube formation of human retinal microvascular endothelial cells (HRMECs) were measured using BrdU, Transwell, and Matrigel assays, respectively. An oxygen-induced retinopathy (OIR) model was induced using neonatal C57BL/6J pups. Anti-miR-155 was intravitreally injected on postnatal day 12, and the retinal non-perfused areas and extent of neovascularization were measured on postnatal day 18 using transcardiovascular fluorescein isothiocyanate (FITC)-dextran perfusion and retina sections. A laser-induced choroidal neovascularization (CNV) model was induced in adult C57BL/6J mice. To evaluate the leakage areas, fundus fluorescein angiography was performed on day 14 after anti-miR-155 intravitreal injection. The neovascularization area of the CNV model was also examined in confocal and retina section studies. The expression levels of SHIP1 and p-Akt (Thr308, Ser473, and Thr450) were evaluated both in vitro and in vivo. RESULTS: The expression of miR-155 was elevated in HRMECs after treatment with vascular endothelial growth factor (VEGF) and in neovascularized mouse model retinas. Anti-miR-155 lentivirus reduced the VEGF-induced proliferation, migration, and tube formation abilities of HRMECs. Anti-miR-155 attenuated retinal neovascularization in in vivo CNV and OIR models. In VEGF-treated HRMECs and retina neovascularization models, p-Akt (Ser473) was significantly upregulated, while SHIP1 was downregulated. Conversely, the inhibition of miR-155 restored the expression of SHIP1 and reduced the phosphorylation of effectors in the Akt (Ser473) signaling pathway. CONCLUSIONS: The results revealed that the downregulation of miR-155 attenuated retinal neovascularization via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.

MicroRNA-185 suppresses growth and invasion of colon cancer cells through inhibition of the hypoxia­inducible factor-2α pathway in vitro and in vivo.

MicroRNAs (miRs) are small non­coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer development and progression. However, the function of miR­185 in the development of human colon cancer has not yet been investigated. In this study, the association between miR­185 expression and the clinicopathological characteristics of patients with colon cancer was analyzed using quantitative polymerase chain reaction (qPCR). Using a gain­of­function approach, the effects of miR­185 overexpression on the expression of hypoxia­inducible factor­2α (HIF­2α), proliferating cell nuclear antigen (PCNA) and matrix metallopeptidase­2 (MMP­2) were investigated in SW620 colon cancer cells using qPCR and western blotting. Functional analysis of cellular proliferative activities, by MTT assay, and invasive potential, by Transwell assay, was conducted on SW620 cells expressing low levels of miR­185. miR­185 was found to be significantly downregulated in cancer tissues compared with adjacent non­cancerous tissues, and was negatively correlated with lymph node metastasis of colon cancer (P<0.001). miR­185 overexpression in vitro impeded cellular proliferation and invasive potential with reduced expression of HIF­2α, PCNA and MMP­2 in SW620 cells transfected with an miR­185 mimic. In addition, the tumor volumes in SW620 subcutaneous nude mouse models treated with miR­185 were significantly smaller than those of the control group. In conclusion, these findings indicate that miR­185 as a tumor suppressor may affect the development of colon cancer cells via inhibition of HIF­2α signaling, suggesting that miR­185 may serve as a potential therapeutic target in cancer treatment.

Efficacy and safety of adalimumab for the Crohn's disease: a systematic review and meta-analysis of published randomized placebo-controlled trials.

PURPOSE: The aim of this study was to evaluate the efficacy and safety of adalimumab (ADA) for Crohn's disease. METHODS: Electronic databases, including PubMed, Embase, the Cochrane Library, and the Science Citation Index, were searched to retrieve relevant trials. We estimated pooled estimates of the odds ratio (OR) and relevant 95% confidence interval (CI) using fixed effects model or random effects model as appropriate. RESULTS: Six randomized placebo-controlled studies met the selection criteria. Short-term clinical response/remission and long-term remission were better in the ADA groups than in the control groups (P < 0.05), both in anti-TNF-naive patients and in subjects who lost their response and/or became intolerant to infliximab (IFX). And ADA was also effective for patients who were previously treated with IFX, and its efficacy in infliximab-exposed patients was probably less than in infliximab-naive patients. In patients with active Crohn's disease (CD), ADA therapy was more effective than placebo for obtaining complete fistula closure. In comparison with placebo, ADA does not increase the risk of serious adverse events. CONCLUSIONS: ADA appears to be effective in achieving short-term clinical response/remission, long-term remission, and complete fistula healing in CD, including patients not manageable with IFX, and appears to have a favorable safety profile. A longer duration of follow-up and a larger number of patients are required to better assess the safety profile of ADA in CD.

Recovery from TNBS-induced colitis leads to the resistance to recurrent colitis and an increased ratio of FOXP3 to CD3 mRNA.

OBJECTIVE: The aim of our study was to investigate the effects of the recovery from acute colitis on recurrent colitis with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. METHODS: Acute colitis was induced via an intrarectal injection of TNBS. For recurrent colitis, mice were intrarectally treated with a repeated TNBS 14 days after the first TNBS administration. And another two groups (control and recovery groups) were added to the analysis. Disease activity index (DAI), macroscopic and histological assessments, mRNA expression of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF), IL-10, forkhead box P3 (FOXP3) and the ratio of FOXP3 to CD3 in the colonic tissues were evaluated. RESULTS: Mice developed colitis after treated with TNBS. After the last TNBS administration, DAI in the recurrent colitis group was lower than that in the acute colitis group. In the recurrent colitis group, the mice exhibited longer colon length, reduced histological damage and lower IL-1ß, IL-6, TNF, IL-10 mRNA expression in the colon compared with the acute colitis group. The ratio of FOXP3 to CD3 mRNA expression in the colon of recurrent colitis was higher than that in the acute colitis. There was a significant negative correlation between the ratio of FOXP3 to CD3 mRNA expression and DAI in the acute and recurrent colitis groups (r= -0.808, P<0.05). CONCLUSIONS: Mice that recovered from TNBS-induced acute colitis with intestinal epithelial disruption are resistant to recurrent colitis, which is associated with an increased ratio of FOXP3 to CD3 mRNA expression.

α-Tocopheryl succinate induces apoptosis in erbB2-expressing breast cancer cell via NF-κB pathway.

AIM: to study the molecular mechanisms underlying α-tocopheryl succinate (α-TOS)-induced apoptosis in erbB2-positive breast cancer cells and to determine whether α-TOS and the human recombinant TNF-related apoptosis-inducing ligand (hrTRAIL) act synergically to induce cell death of erbB2-expressing breast cancer cells. METHODS: the annexin V binding method was used to measure apoptosis induced by α-TOS and/or hrTRAIL. RT-PCR and Western blotting were performed to detect gene and protein expression. A colorimetric assay was performed to detect caspase activity. The TransAM(TM) NF-κB p65 kit was used to assess NF-κB activation. RESULTS: α-TOS (100 µmol/L) significantly inhibited NF-κB nuclear translocation in erbB2-expressing breast cancer cells; this inhibition is expected to result in the inactivation of NF-κB. α-TOS (50 and 100 µmol/L) inhibited the expression of Flice-like inhibitory protein (FLIP) and cellular inhibitor of apoptosis protein 1 (c-IAP1) in erbB2-positive cells. α-TOS (100 µmol/L) inhibited Akt activation and augmented the activity of caspase 3 and caspase 8 in breast cancer cells expressing erbB2. α-TOS (50 µmol/L) and hrTRAIL (30 mg/mL) acted synergically to induce apoptosis in breast cancer cells. α-TOS also decreased the hrTRAIL-induced transient activation of NF-κB . CONCLUSION: our results suggest that α-TOS mediates the apoptosis of erbB2-positive breast cancer cells and acts synergically with hrTRAIL via the NF-κB pathway.

[Anti-tumor effect of monkshood polysaccharide with adriamycin long circulating temperature-sensitive liposome and its mechanism].

AIM: To observe the synergic action of monkshood polysaccharide (MPS) and adriamycin (ADM) long circulating temperature-sensitive liposome (ALTSL) in targeting therapy for H22 tumor-bearing mice and explore the mechanism. METHODS: The anti-tumor activity was evaluated by using the tumor's weight as an index. The life prolongation rate of mice was calculated according to the survival time of the tumor-bearing mice. The killer activity of NK cells and the lymphocyte transformation rate were detected by the LDH release assay and MTT colorimetry, respectively. The apoptosis of tumor cells and the expressions of p53, Fas, Fas-L and caspase-3 were analyzed by flow cytometry. The expressions of IL-2 mRNA and IL-12 mRNA in spleen lymphocytes were determined by RT-PCR. The pathologic changes of tumor, heart, liver and kidney tissues of the tumor-bearing mice were observed under light microscope. RESULTS: Compared with the adriamycin liposome group, the anti-tumor effects were enhanced in (MPS+ALTSL) group with tumor growth inhibitory rate up to 80.4%. The survival time of the tumor-bearing mice in ALTSL and (MPS+ALTSL) groups was significantly prolonged compared with the ADM group (P<0.01). The killer activity of NK cells was higher in ALTSL group than in the NS and ADM groups, and was highest in (MPS+ALTSL) group. The lymphocyte transformation rate of (MPS+ALTSL) group was markedly increased (P<0.01) as compared with the ADM group. The result of RT-PCR indicated that the expressions of IL-2 mRNA and IL-12 mRNA in lymphocytes in the adriamycin long circulating liposome (ALCL) group were significantly higher than those in the ADM group. Expressions of IL-2 mRNA and IL-12 mRNA was much higher in (MPS+ALTSL) group than in ALTSL group. The pathological examination indicated that in (MPS+ALTSL) group, more lymphocytes and monocytes were found in tumor tissue. CONCLUSION: ALTSL can increase the anti-tumor effect and decrease the side-effects (such as the cytotoxicity) of ADM. MPS combined with ALTSL can enhance killer activity of NK cells and transformation of T cells, supporting their synergic anti-tumor effect.

[Synergistic role between rhIL-2 and adriamycin long circulating temperature-sensitive liposome in targeting therapy on tumor].

AIM: To observe the synergistic role between rhIL-2 and adriamycin long circulating temperature-sensitive liposome (ALTSL) in targeting therapy of H22 tumor-bearing mice and explore their anti-tumor mechanism. METHODS: The antitumor activity was evaluated by using the tumor's weight as an index. The prolongation rate of mouse life was calculated according to the survival time of the tumor-bearing mice. The killer activity of NK cells and the lymphocyte transformation rate were detected by the LDH and MTT colorimetry, respectively. The apoptosis of tumor cells and the expression of p53, Fas, Fas-L and Caspase-3 were analyzed by flow cytometry (FCM). The expression of IL-2 mRNA and IL-12 mRNA in splenocytes was determined by RT-PCR. The pathologic changes of tumor, heart, liver and kidney tissues of the tumor-bearing mice were observed under light microscope. RESULTS: The tumoristatic rate of rhIL-2+ALTSL (73.5%) was higher than that of adriamycin liposome (ADML) group (67.0%). The survival time of tumor-bearing mice in ALTSL and rhIL-2+ALTSL groups was significantly extended as compared with the NS group (treated with normal saline) and the free ADM group (P <0.01 or P <0.05). The killer activities of NK cells of ALTSL group and rhIL-2+ALTSL group were higher than those of the NS and free ADM groups, and was highest in rhIL-2+ALTSL group. The lymphocyte transformation rate of ALTSL+rhIL-2 group markedly increased ( P <0.01) as compared with the free ADM group. The result of RT-PCR indicated that the expression of IL-2 mRNA and IL-12 mRNA in splenocytes in the adriamycin long circulating liposome (ALCL) group was significantly higher than that in the free ADM group. The enhancement of rhIL-2+ALTSL on expression of IL-2 mRNA and IL-12 mRNA was much stronger than that of ALTSL alone. The pathological examination indicated that in rhIL-2+ALTSL group, the tumor cells were mostly destroyed, and a large amount of lymphocytes and monocytes were found in tumor tissue. CONCLUSION: ALTSL can increase the anti-tumor effect and decreased the side-effects (such as the cytotoxicity) of ADM. rhIL-2+ALTSL can induce the apoptosis of tumor cells and enhance killer activities of T cells and NK cells. rhIL-2 and ALTSL can synergistically play the antitumor effect.

[Preparation of monoclonal antibody against human c-erbB2 and identification of its specificity].

AIM: To prepare monoclonal antibody(mAb) against human c-erbB2 and identify its specificity. METHODS: The epitope of human c-erbB2 antigen was analyzed by using computer software and a immunodominant epitope at the carboxyl-terminal was selected. A peptide consisting of 13 amino acids was synthesized and coupled with keyholelimpet hemocyanin (KLH), and then it was used to immunize BLAB/c mice. The splenocytes of the immunized mice were fused with Sp2/0 cells routinely and the hybridoma cells were selected by HAT selected culture, indirect ELISA, and immunohistochemical staining, and cloned by limiting dilution. The specificity of the mAb was identified by cross-reaction test and blocking test. RESULTS: A hybridoma cell line SC8C1, stably secreting anti-c-erbB2 mAb was obtained. The mAb SC8C1 could react to breast cancer tissue expressing c-erbB2 molecule but did not react to other c-erbB2-negative cells. The mAb will lose the activity after being blocked with synthesized 13 peptide. CONCLUSION: A anti-c-erbB2 mAb SC8C1 is prepared successfully using synthesized 13 peptide as immunogen.

[The effects of two TCM prescriptions, Tonifying Kidney and Strengthening Vital Energy, on NF-kappaB activing in aged mouse T cells].

AIM: The aim of the present study was to explore the immunologic mechanism of delaying senescence by Strengthening Vital Energy(SVE), Tonifying Kidney (TK) and combined TK and SVE. METHODS: Mice of 20 months were used as senescence model. The effects of the prescriptions on anti-CD3 antibody-induced NF-kappaB activity and the expression of NF-kappaB in T cells from aged mice were analyzed by electrophoretic mobility shift assay (EMSA) and Western blot, respectively. RESULTS: NF-kappaB activity in anti-CD3 antibody-induced T cells from the aged mice was lower than that from young ones. The three prescriptions raised the activity of NF-kappaB in the T cells from the aged mice to certain extent. Combined TK and SVE had no influence on p65 and p50 subunits expressions. CONCLUSION: The increased NF-kappaB activity may be one of mechanisms underlying the improvement of immune response in aged mice by Chinese medicines.

[Effect of thalidomide on tumor growth in mouse hepatoma H22 model].

BACKGROUND & OBJECTIVE: Thalidomide may have curative effect on tumor because of its function of anti-angiogenesis. The aim of this research was to observe the effect of thalidomide on tumor homograft growth in mouse H22 model and to investigate the mechanisms involved and its curative possibility to hepatoma. METHODS: BALB/c mice were inoculated subcutaneously with H22 cells. The first treatment group was injected intraperitoneal everyday with thalidomide 50 mg/kg from the day of inoculation and the second treatment group was injected from the fourth day of inoculation. The diameters of the tumors were measured everyday. On the twelfth day, the mice were sacrificed and the tumors were weighed. The microvessel densities of the tumors were measured by immunohistochemical staining with anti-CD31 monoclonal antibody; CD31 expression, apoptosis, and proliferation were analyzed by flow cytometry. Expression of vascular endothelial growth factor (VEGF) mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The tumor weight of the first treatment group (2.27+/-0.33 g) was decreased compared with that of control group (2.70+/-0.61 g) (P >0.05); the ratio of inhibition was 15.93%. The tumor weight of the second treatment group (3.32+/-0.47 g) was decreased compared with that of control group (3.42+/-0.60 g) (P >0.05); the ratio of inhibition was 2.92%. The microvessel count (48.40+/-12.90) and CD31 expression (5.59+/-0.75) of the first treatment group were significantly decreased (P< 0.05) in comparison with those of control groups (81.20+/-26.91,7.04+/-0.50) (P< 0.05). The microvessel count (46.4+/-9.71) and CD31 expression (7.29+/-0.48) of the second treatment group were not statistically significant (P >0.05) compared with those of control groups (74.0+/-32.69, 6.80+/-0.68) (P >0.05). The apoptosis indices of the two treatment groups (17.20+/-7.80,15.33+/-4.10) were significantly increased, compared with control groups (4.37+/-1.98,4.87+/-1.91) (P< 0.05), while there was no significant difference of VEGF mRNA expression between the two treatment groups and control groups (P >0.05). The relative quantities of VEGF mRNA of the two treatment groups were 0.50+/-0.13 and 0.51+/-0.06 (control groups:0.48+/-0.11 and 0.64+/-0.11) (P >0.05), respectively. CONCLUSION: Thalidomide can significantly induce apoptosis and inhibit angiogenesis in mouse H22 model when it is used at the beginning of carcinogenesis, but it has no obvious anti-angiogenic efficacy on the grown tumor. Thalidomide cannot inhibit VEGF mRNA expression of grafted H22 tumor in mouse.

[Cooperative effect of rhIL-2 and adriamycin-magnetic albumin microsphere targeting therapy on tumor].

AIM: To observe the synergistic inhibitory effects of rhIL-2 and adriamycin magnetic albumin microsphere(ADM-MAM) targeting therapy on tumor and to explore their antitumor mechanism. METHODS: The antitumor activity was observed using the tumor weight as index. The killer activity of natural killer (NK) cells and the lymphocyte transformation were examined by the LDH release assay and MTT colorimetry, respectively. The apoptosis of tumor cells and the expressions of p53,Fas and FasL were examined by flow cytometry. The expressions of IL-2 and IL-12 were determined by RT-PCR. RESULTS: Compared with the control group, in ADM-MAM targeting therapy group and combination therapy (ADM-MAM and rhIL-2 therapy) group, tumor weight, killer activity of NK cells, and the lymphocyte transformation;were obviously decreased; while the expression of Fas, FasL, IL-2 and IL-12 were markedly increased. CONCLUSION: ADM-MAM targeting therapy exerts anti-tumor effects with fewer side effects, which can be enhanced when combined with rhIL-2 therapy. Such synergistic anti-tumor effects may be realized by stimulating the proliferation of T cells and growth of NK cell to enhance cellular immunological function.