OBJECTIVE: To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats. METHODS: Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 μmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors-transforming growth factor β ₁ (TGF-β ₁), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type Ⅱ. RESULTS: The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L ( P<0.05), so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-β ₁, IL-6, and TNF-α in cells significantly increased when compared with the control group ( P<0.05). However, intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group ( P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 ( P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type Ⅱ expression. CONCLUSION VX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.
Rats ; Animals ; Chondrocytes/metabolism* ; Matrix Metalloproteinase 13/metabolism* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha/metabolism* ; Collagen Type II/metabolism* ; Interleukin-6 ; Lipopolysaccharides/pharmacology* ; NF-E2-Related Factor 2/pharmacology* ; Inflammation/drug therapy* ; Osteoarthritis/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Dipeptides ; para-Aminobenzoates

Romipeptides A and B (1 and 2), two new romidepsin derivatives, and three known compounds, chromopeptide A (3), romidepsin (4) and valine-leucine dipeptide (5) were isolated from the fermentation broth of Chromobacterium violaceum No. 968. Their structures were elucidated by interpretation of their UV, HR-ESI-MS and NMR spectra. The absolute configuration of compound 1 and 2 were established by single crystal X-ray diffraction analysis. Compounds 1-5 were evaluated for their anti-proliferative activities against three human cancer cell lines, SW620, HL60, and A549. The results showed most of these compounds exhibited antitumor activities in vitro, in which compound 2 displayed potent cytotoxicity to SW620, HL60 and A549 cell lines, with IC of 12.5, 6.7 and 5.7 nmol·L, respectively.
Antineoplastic Agents ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Chemistry Techniques, Analytical ; Chromobacterium ; metabolism ; Depsipeptides ; chemistry ; pharmacology ; Dipeptides ; chemistry ; Drug Screening Assays, Antitumor ; Fermentation ; Humans ; Molecular Structure ; Peptides, Cyclic ; chemistry

Two new isomeric modified tripeptides, aspergillamides C and D (compounds 1 and 2), together with fifteen known compounds (compounds 3-17), were obtained from the marine sponge-derived fungus Aspergillus terreus SCSIO 41008. The structures of the new compounds, including absolute configurations, were determined by extensive analyses of spectroscopic data (NMR, MS, UV, and IR) and comparisons between the calculated and experimental electronic circular dichroism (ECD) spectra. Butyrolactone I (compound 11) exhibited strong inhibitory effects against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) with the IC being 5.11 ± 0.53 μmol·L, and acted as a noncompetitive inhibitor based on kinetic analysis.
4-Butyrolactone ; analogs & derivatives ; chemistry ; isolation & purification ; pharmacology ; Animals ; Aspergillus ; chemistry ; Chemistry Techniques, Analytical ; Dipeptides ; chemistry ; isolation & purification ; pharmacology ; Enzyme Inhibitors ; chemistry ; isolation & purification ; pharmacology ; Indoles ; chemistry ; isolation & purification ; pharmacology ; Molecular Structure ; Mycobacterium tuberculosis ; drug effects ; Peptides ; chemistry ; isolation & purification ; pharmacology ; Polyketides ; chemistry ; isolation & purification ; pharmacology ; Porifera ; microbiology ; Protein Tyrosine Phosphatases ; chemistry

OBJECTIVE: To investigate the role of cathepsin B in hepatic Kupffer cells (KCs) in activating Toll-like receptor 4(TLR- 4)-independent inflammatory pathways in mice with lipopolysaccharide (LPS)-induced sepsis. METHODS: Eighteen wild-type (WT) mice and 18 TLR4-knockout (TLR4) mice were both divided into 3 groups for intraperitoneal injections of a lethal dose (54 mg/kg) of LPS, LPS and CA-074(a cathepsin B inhibitor), or normal saline, and the survival of the mice were observed. Another 36 WT mice and 36 TLR4mice were also divided into 3 groups and subjected to intraperitoneal injections of normal saline, 20 mg/kg LPS, or LPS with CA-074 pretreatment.After the treatments, KCs were collected from the mice for assessing the protein level and activity of cathepsin B.The histopathological changes of the liver were observed with HE staining, and the serum levels of IL-1α, IL-1β, TNF-α and IL-18 were detected. RESULTS: Compared with the WT mice,TLR4mice receiving the lethal dose of LPS had significantly longer survival time (up to 84 h) after the injection,but were still unable to fully resist LPS challenge.CA-074 pretreatment prolonged the survival time of WT mice and TLR4mice to 60 h and 132 h,respectively.In the mouse models of sepsis,20 mg/kg LPS induced significantly enhanced activity of cathepsin B without affecting its expression level in the KCs (<0.05) and increased the serum levels of the inflammatory cytokines.CA-074 pretreatment of the mice obviously lessened the detrimental effects of LPS in TLR4mice by significantly lowering cathepsin B activity in the KCs,alleviating hepatocyte apoptosis and reducing the serum levels of inflammatory cytokines. CONCLUSIONS Cathepsin B plays an important role in activating TLR4-independent inflammatory pathways in mice with LPS-induced sepsis.
Animals ; Cathepsin B ; antagonists & inhibitors ; physiology ; Dipeptides ; pharmacology ; Gene Knockout Techniques ; Hepatocytes ; Inflammation ; metabolism ; Interleukin-18 ; blood ; Interleukin-1alpha ; blood ; Interleukin-1beta ; blood ; Kupffer Cells ; metabolism ; Lipopolysaccharides ; Liver ; pathology ; Mice ; Sepsis ; etiology ; metabolism ; Toll-Like Receptor 4 ; genetics ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo investigate the effect of γ-secretase inhibitor (N-[N-(3,5-difluorophenacetyl)-l -alanyl]-S-phenylglycine t-butyl ester, DAPT) on hyperoxia-induced brain white matter injury in mice.

MWTHODSThree-day-old C57BL/10J mouse pups were divided into air control (C) group, control+DAPT (10 mg/kg, injected intraperitoneally) group, hyperoxia group (exposed to 80% oxygen for 48 h), and hyperoxia+DAPT group. The brain and body weights of the mice were measured at postnatal days 3, 5, 12, and 28. Real-time PCR was used to detect Notch intracellular domain (NICD) mRNA expression in the brain after modeling, and the expressions of NG2 and myelin basic protein (MBP) were detected by double-labeled immunofluorescence assay to verify the oligdendrocycle type at postnatal day 12. The mice in each group were bred until postnatal day 28 for Morris water maze test.

RESULTSThe brain and body weights were significantly decreased in mice in hyperoxia group compared to the control mice, but increased significantly after DAPT treatment (P<0.05). Real-time PCR showed that a 48-hour hyperoxia exposure significantly increased NICD mRNA expression in the brain (P<0.05), which was decreased by co-treatment by DAPT (P<0.05). Hyperoxia also resulted in enhanced NG2 expression and lowered MBP expression in the brain (P<0.05). Compared with the control mice, the mice exposed to hyperoxia showed prolonged escape latency (P<0.05) and spent less time in the target quadrant with a lowered number of passing through the virtual platform (P<0.05). All these parameters were significantly improved by co-treatment with DAPT.

CONCLUSIONSpecific inhibition of Notch signaling pathway activation in the brain by the γ-secretase inhibitor DAPT can ameliorate white matter injury and learning and memory impairment in newborn mice with hyperoxia exposure.

Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; Animals ; Body Weight ; Brain ; metabolism ; pathology ; Dipeptides ; pharmacology ; Hyperoxia ; physiopathology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Organ Size ; Receptors, Notch ; metabolism ; Signal Transduction ; White Matter ; pathology

This study aims to investigate the effect of γ-Secretase Inhibitor DAPT, (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), on the differentiation of neural precursor cells and the production of neurons in the neural precursor cell line GE6. GE6 was cultured in medium with 4 μmol/L DAPT added as the experimental group and the untreated medium separately as the control group. After 4 days of differentiation, we carried out the following experiments. We used immuno-fluorescent staining to observe the ratio of Tuj1, GFAP and O4 positive cells. We also used qRT-PCR to detect the effect of the DAPT on Tuj1 and GFAP mRNA transcription in the GE6. The results of immuno-fluorescent staining indicated that the Tuj1 ratio of experimental group was higher compared to that of the control group, but the GFAP and O4 ratio of experimental group was lower than that of the control group. The differences were statistically significant (P < 0.05). The result of qRT-PCR was in accordance with immunofluorescent staining results. It was well concluded that DAPT could promote the neurogenic differentiation of neural precursor cell line rather than leading to gliogenic differentiation. More neurons could be obtained for transplantation with the addition of DAPT.
Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; Animals ; Cell Differentiation ; drug effects ; Cell Line ; Dipeptides ; pharmacology ; Neural Stem Cells ; drug effects ; Neurons ; Rats

OBJECTIVETo study the effect of L-alanyl-L-glutamine (Ala-Gln) on the levels of insulin-like growth factor-1 (IGF-1) and IGF-1 receptor (IGF-1R) in the intestinal tissues of low-birth-weight (LBW) newborn rats with hypoxia/reoxygenation-induced intestinal injury.

METHODSPregnant rats were fed with or without smoking. The rats born by those fed without smoking were included in group A; for the rats born by those fed with smoking, normal-birth-weight rats were included in group B, and LBW rats were randomly divided into control group (group C), hypoxia/reoxygenation (H/R) group (group D), and Ala-Gln group (group E). Each group consisted of 24 newborn rats. The rats in groups D and E received H/R treatment twice a day for three consecutive days to establish an intestinal injury model; the rats in group E were intraperitoneally injected with Ala-Gln (10 ml/kg) before daily H/R treatment, while those in groups C and D were given an equal dose of normal saline by intraperitoneal injections. On days 4, 7, and 10 after birth, 8 rats were sacrificed in each group to collect intestinal tissues. The IGF-1 levels in intestinal tissues were measured using ELISA, and IGF-1R levels were measured by immunohistochemistry.

RESULTSThere were no significant differences in IGF-1 and IGF-1R levels between groups A and B at all time points. The levels of IGF-1 and IGF-1R in group C kept increasing, were higher than those in other groups on day 7 (P<0.05), and reached a normal level on day 10, without significant differences compared with those in groups A and B. Group D had significantly lower IGF-1 and IGF-1R levels than group C at all time points (P<0.05). The levels of IGF-1 and IGF-1R in group E were lower than those in group C on days 4 and 7 (P<0.05), but they increased to approximately the levels in group C and were significantly higher than those in group D on day 10.

CONCLUSIONSIntrauterine and postnatal hypoxia may induce intestinal injury in LBW newborn rats, and parenteral administration of high-dose Ala-Gln can reduce hypoxia-induced intestinal injury. Therefore, Ala-Gln has a protective effect against hypoxia-induced intestinal injury.

Animals ; Birth Weight ; Dipeptides ; pharmacology ; Female ; Hypoxia ; metabolism ; Insulin-Like Growth Factor I ; analysis ; Intestines ; chemistry ; Male ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Receptor, IGF Type 1 ; analysis

BACKGROUNDPancreatic cancer is a common malignant tumor of the digestive system. It is the fourth major cause of tumor-related death and its morbidity is increasing, and hence it is imperative to develop effective forms of therapy for pancreatic cancer. Peptide tyrosine-tyrosine (PYY) is an important gastrointestinal peptide hormone. According to previous literatures, PYY has been shown to inhibit tumor proliferation in cellular and animal models, but there has been limited research on the detailed mechanism of PYY in pancreatic cancer. This study was to observe the effects of PYY on pancreatic cancer cell and investigate the possible mechanism.

METHODSThe expression of Y1, Y2, and Y5 receptors on pancreatic cancer cell lines were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The cytotoxicity of PYY toward the MiaPaCa-2 cell was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; the cell morphology and structure changes were observed under inverted microscope and transmission electron microscope respectively. Apoptosis and cell cycle were evaluated by flow cytometry. The activity of caspase-3 was determined by activity assay kits and Western blotting. The expression of survivin, vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2) were determined by RT-qPCR and Western blotting.

RESULTSExpression of Y2 receptor is the most abundant PYY receptor on pancreatic cancer cell. PYY inhibited MiaPaCa-2 cell proliferation, blocked it in G0/G1 phase, increased the proportion of apoptosis cells and caspase-3 activity, and reduced the expression of survivin, VEGF, and COX-2.

CONCLUSIONSPYY weakened the ability of the pancreatic MiaPaCa-2 cell viability through cell cycle blocking and apoptosis inducing. The inhibition effect of PYY may be mediated by the Y2 receptor. The increased caspase-3 activity and reduced expression of survivin, VEGF, and COX-2 may serve as a novel mechanism in PYY inhibition effect on MiaPaCa-2 cell.

Blotting, Western ; Caspase 3 ; genetics ; metabolism ; Cell Cycle ; drug effects ; genetics ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 ; genetics ; metabolism ; Dipeptides ; pharmacology ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Pancreatic Neoplasms ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo study the effect of matrix metalloproteinases inhibitor GM6001 in suppressing scar tissue formation in the filtering passage after glaucoma filtration surgery.

METHODSTwenty-four pigmented rabbits (48 eyes) underwent trabeculectomy followed by subconjunctival injection of GM6001 in the right eye (treated eyes) and injection of PBS in the left eye (control) once a day. The intraocular pressure was monitored postoperatively and proliferating cell nuclear antigen (PCNA)- and α-smooth muscle actin (α-SMA)-positive cells in the filtering pathway were detected using immunohistochemistry.

RESULTSOn postoperative days 7, 14, 21, and 28, the intraocular pressure was significantly lower in the treated eyes (GM6001) than in the control eyes (P<0.01). The counts of PCNA- and α-SMA-positive cells were also significantly lowered in the treated than in the control eyes (P<0.01).

CONCLUSIONGM6001 can inhibit excessive proliferation of the fibroblasts in the filtering pathway to suppress scar tissue formation and prolong the existence of the functional filtration bleb in rabbits.

Actins ; metabolism ; Animals ; Cicatrix ; pathology ; prevention & control ; Dipeptides ; pharmacology ; Filtering Surgery ; adverse effects ; Glaucoma ; surgery ; Intraocular Pressure ; Postoperative Complications ; Proliferating Cell Nuclear Antigen ; metabolism ; Rabbits

BACKGROUNDPaclitaxel (PAC) is the first-line chemotherapy drug for most breast cancer patients, but clinical studies showed that some breast cancer patients were insensitive to PAC, which led to chemotherapy failure. It was reported that Notch1 signaling participated in drug resistance of breast cancer. Here, we show whether Notch1 expression is related to PAC sensitivity of breast cancer.

METHODSWe employed Notch1 siRNA and Notch1 inhibitor, N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester (DAPT), to down regulate Notch1 expression in human breast cancer cells MDA-MB-231, and detected the inhibition effect by Western blotting and reverse trans cription-polymerase chain reaction, respectively. After 24 hours exposure to different concentration of PAC (0, 1, 5, 10, 15, 20, and 25 µg/ml), the viability of the control group and experimental group cells was tested by MTT. We also examined the expression of Notch1 in PAC sensitive and nonsensitive breast cancer patients, respectively by immunohistochemistry (IHC). The PAC sensitivity of breast cancer patients were identified by collagen gel droplet embedded culture-drug sensitivity test (CD-DST).

RESULTSDown regulation of Notch1 expression by Notch1siRNA interference or Notch1 inhibitor increased the PAC sensitivity in MDA-MB-231 cells (P < 0.05). Also, the expression of Notch1 in PAC sensitive patients was much lower than that of PAC non-sensitive patients (P < 0.01).

CONCLUSIONNotch1 expression has an effect on PAC sensitivity in breast cancer patients, and the inhibition of Notch1 increases paclitaxel sensitivity to human breast cancer.

Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dipeptides ; pharmacology ; Humans ; Immunohistochemistry ; Paclitaxel ; pharmacology ; Receptor, Notch1 ; antagonists & inhibitors ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction