OBJECTIVETo investigate the antiviral activity of 3-hydroxyphthalic anhydride-modified ovalbumin (HP-OVA) against herpes simplex virus 2 (HSV-2) in vitro.

METHODSBy chemical modification, ovalbumin (OVA) was treated with 3-hydroxyphthalic anhydride (HP) to prepare HP-OVA. The anti-HSV-2 activity against HSV-2 333 virus in vitro and the cytotoxicity of HP-OVA in African green monkey kidney cells (Vero cells) were detected by MTT colorimetric assay. The inhibitory effects of HP-OVA on 17 strains of vaginal lactobacilli were observed by microscopy.

RESULTSAnhydride-modified ovalbumin significantly inhibited the infection by HSV-2 with an IC(50) of 23.56±8.33 µg/ml. HP-OVA showed only low cytotoxicity to the host cells with a CC(50) over 1 mg/ml. HP-OVA did not produce significant inhibitory effect on the 17 strains of vaginal lactobacilli (MIC>1 mg/ml).

CONCLUSIONAnhydride-modified protein HP-OVA exhibits potent anti-HSV-2 activity in vitro and can be a good microbicide candidate for prevention of sexually transmitted diseases.

Animals ; Antiviral Agents ; pharmacology ; Cercopithecus aethiops ; Herpesvirus 2, Human ; drug effects ; Ovalbumin ; chemistry ; pharmacology ; Phthalic Anhydrides ; chemistry ; pharmacology ; Vero Cells

Two new 2-carboxymethyl-3-hexyl-maleic anhydride derivatives, arthrianhydride A (1) and B (2), along with three known compounds 3-5, were isolated from the fermentation broth of a grasshopper-associated fungus Arthrinium sp. NF2410. The structures of new compounds 1 and 2 were determined based on the analysis of the HR-ESI-MS and NMR spectroscopic data. Furthermore, compounds 1 and 2 were evaluated on inhibitory activity against the enzyme SHP2 and both of them showed moderate inhibitory activity against SHP2.
Anhydrides/pharmacology* ; Animals ; Biological Products/pharmacology* ; Enzyme Inhibitors/pharmacology* ; Fungi/chemistry* ; Grasshoppers/microbiology* ; Molecular Structure ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors* ; Secondary Metabolism

Objective: To investigate the effects of methacrylic anhydride gelatin (GelMA) hydrogel loaded with silver and recombinant human basic fibroblast growth factor (rh-bFGF) on deep partial-thickness burn wounds in rabbits. Methods: The experimental research method was adopted. Low-concentration GelMA materials, medium-concentration GelMA materials and high-concentration GelMA materials containing different concentrations of methacrylic anhydride (MA) were prepared, after adding photoinitiator, low-concentration GelMA hydrogels, medium-concentration GelMA hydrogels, and high-concentration GelMA hydrogels were obtained, respectively. The nuclear magnetic resonance spectroscopy was performed to detect the hydrogen nuclear magnetic resonance spectra of the above-mentioned three concentrations of GelMA materials, and to calculate the degree of substitution according to the spectrum diagram. The three-dimensional microstructure and pore size of 3 types of above-mentioned GelMA hydrogels were detected by field emission scanning electron microscopy (FESEM), with 9 samples measured. According to the selected concentration of MA, ten kinds of solutions of GelMA with different concentration of silver (silver-containing GelMA) were synthesized, and the silver-containing GelMA solution of each concentration was divided into three parts, and then exposed to ultraviolet light lasting for 20, 25, and 35 s, respectively. After adding photoinitiator,the corresponding silver-containing GelMA hydrogels were obtained. The residual degradation rate of silver-containing GelMA hydrogel with different photocrosslinking times was detected by collagenase degradation method at degradation of 12, 24, 36, and 48 h; and the time required for complete degradation was detected, and the sample number was 5. The inhibition zone diameter of GelMA hydrogel under above screened photocrosslinking times containing 10 concentrations of silver against Staphylococcus aureus was measured to reflect its antibacterial ability, and the sample numbers were all 5. The silver-containing GelMA hydrogel with statistical significance compared with the antibacterial circle diameter of the silver-containing GelMA hydrogel containing the lowest concentration (no silver) was considered as having antibacterial activity. The three-dimensional microstructure and pore size of the silver-containing GelMA hydrogels with antibacterial activity and the lowest drug concentration selected were detected by FESEM, and the sample numbers were all 9. The freeze-dried alone GelMA hydrogel and the freeze-dried silver-containing GelMA hydrogel were soaked in phosphate buffer solution for 24 h, respectively, then the swelling rate of the two GelMA hydrogel were calculated and compared by weighing method, and the sample number was 5. GelMA hydrogel containing silver and rh-bFGF, namely compound hydrogel for short, was prepared according to the preliminary experiment and the above experimental results. The appearance of the composite hydrogel was observed in general, and its three-dimensional microstructure and pore size were detected by FESEM. The deep partial-thickness burn wound was made on the back of 30 rabbits (aged 4-6 months, female half and half). Meanwhile, with the rabbit head as the benchmark, the wounds on the left side of the spine were treated as composite hydrogel treatment group, and the wounds on the right side were treated as gauze control group, and which were treated accordingly. On post injury day (PID) 3, 7, 14, 21, and 28, the healing of wounds in the two groups was observed. On PID 7, 14, 21, and 28, the wound healing area was recorded and the healing rate was calculated, with a sample number of 30. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: The substitution degree among low-concentration GelMA materials, medium-concentration GelMA materials, and high-concentration GelMA materials was significantly different (F=1 628.00, P<0.01). The low-concentration GelMA hydrogel had a loose and irregular three-dimensional spatial network structure with a pore size of (60±17) μm; the medium-concentration GelMA hydrogel had a relatively uniform three-dimensional spatial network and pore size with a pore size of (45±13) μm; the high-concentration GelMA hydrogel had the dense and disordered three-dimensional spatial network with a pore size of (25±15) μm, the pore sizes of 3 types of GelMA hydrogels were significantly differences (F=12.20, P<0.01), and medium concentration of MA was selected for the concentration of subsequent materials. The degradability of silver-containing GelMA hydrogels with different concentrations of the same photocrosslinking time was basically same. The degradation residual rates of silver-containing GelMA hydrogels with 20, 25, and 35 s crosslinking time at 12 h were (74.2±1.7)%, (85.3±0.9)%, and (93.2±1.2)%, respectively; the residual rates of degradation at 24 h were (58.3±2.1)%, (65.2±1.8)%, and (81.4±2.6)%, respectively; the residual rates of degradation at 36 h were (22.4±1.9)%, (45.2±1.7)%, and (68.1±1.4)%, respectively; the residual rates of degradation at 48 h were (8.2±1.7)%, (32.4±1.3)%, and (54.3±2.2)%, respectively, and 20, 25, and 30 s photocrosslinking time required for complete degradation of silver-containing GelMA hydrogels were (50.2±2.4), (62.4±1.4), and (72.2±3.2) h, and the difference was statistically significant (F=182.40, P<0.01), 25 s were selected as the subsequent photocrosslinking time. The antibacterial diameters of 10 types of silver-containing GelMA hydrogels against Staphylococcus aureus from low to high concentrations were (2.6±0.4), (2.5±0.4), (3.2±0.4), (12.1±0.7), (14.8±0.7), (15.1±0.5), (16.2±0.6), (16.7±0.5), (16.7±0.4), and (16.7±0.6) mm, respectively, and which basically showed a concentration-dependent increasing trend, and the overall difference was statistically significant (F=428.70, P<0.01). Compared with the silver-containing GelMA hydrogel with the lowest concentration, the antibacterial circle diameters of other silver-containing GelMA hydrogels with antibacterial ability from low to high concentration were significantly increased (with t values of 26.35, 33.84, 43.65, 42.17, 49.24, 55.74, and 43.72, respectively, P<0.01). The silver-containing GelMA hydrogel with the antibacterial diameter of (12.1±0.7) mm had the lowest antibacterial activity against Staphylococcus aureus and the lowest drug loading concentration, and the concentration of silver was selected for the concentration of subsequent materials. The microscopic morphology of the silver-containing GelMA hydrogel containing silver element with a pore size of (45±13) μm had a regular and linear strip-like structure. After soaking for 24 h, the swelling ratio of silver-containing GelMA hydrogel was similar to that of alone GelMA hydrogel. The composite hydrogel was colorless, clear and transparent, and its three-dimensional microstructure was a regular and uniform grid, with a filament network structure inside, and the pore size of (40±21) μm. On PID 3, a large amount of necrotic tissue and exudate of rabbit wound in composite hydrogel group were observed, and scattered scabs, a small amount of necrotic tissue and exudate of rabbit wound in gauze control group were observed. On PID 7, the area of rabbit wound in composite hydrogel group was significantly reduced, and adhesion of rabbit wound and gauze in gauze control group was observed. On PID 14, In composite hydrogel group, the rabbit wound surface was ruddy, and the growth of granulation tissue was observed, and in gauze control group, the rabbit wound base was pale, and the blood supply was poor. On PID 21, the rabbit wounds in composite hydrogel group healed completely, and rabbit wound in gauze control group had healing trend. On PID 28, new hair could be seen on rabbit wound surface in composite hydrogel group; oval wound of rabbit in gauze control group still remained. On PID 7, 14, 21, and 28, the wound healing areas of rabbit in composite hydrogel group were significantly larger than those in gauze control group (with t values of 2.24, 4.43, 7.67, and 7.69, respectively, P<0.05 or P<0.01). Conclusions: The medium-concentration GelMA hydrogel has good physical and chemical properties in terms of swelling and degradability. The screened silver-containing GelMA hydrogels had the lowest antibacterial activity and the lowest drug loading concentration. Composite hydrogel can significantly shorten the healing time of deep partial-thickness burn wounds in rabbits.
Anhydrides ; Animals ; Anti-Bacterial Agents ; Burns/drug therapy* ; Female ; Fibroblast Growth Factor 2 ; Gelatin/pharmacology* ; Humans ; Hydrogels/pharmacology* ; Rabbits ; Recombinant Proteins ; Staphylococcal Infections ; Staphylococcus aureus

Objective: To prepare graphene oxide (GO)-containing gelatin methacrylate anhydride (GelMA) hydrogel and to investigate the effects of in situ photopolymerized GO-GelMA composite hydrogel in wound vascularization of full-thickness skin defect in mice. Methods: The experimental study method was used. The 50 μL of 0.2 mg/mL GO solution was evenly applied onto the conductive gel, and the structure and size of GO were observed under field emission scanning electron microscope after drying. Human skin fibroblasts (HSFs) were divided into 0 μg/mL GO (without GO solution, the same as below) group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, 5.0 μg/mL GO group, and 10.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the absorbance value was detected using a microplate analyzer after 48 h of culture to reflect the proliferation activity of cells (n=6). HSFs and human umbilical vein vascular endothelial cells (HUVECs) were divided into 0 μg/mL GO group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the migration rates of HSFs at 24 and 36 h after scratching (n=5) and HUVECs at 12 h after scratching (n=3) were detected by scratch test, and the level of vascular endothelial growth factor (VEGF) secreted by HSFs after 4, 6, and 8 h of culture was detected by enzyme-linked immunosorbent assay method (n=3). The prepared GO-GelMA composite hydrogels containing GO of the corresponding final mass concentration were set as 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group to observe their properties before and after cross-linking, and to detect the release of GO after soaking with phosphate buffer solution for 3 and 7 d (n=3). The full-thickness skin defect wounds were made on the back of 16 6-week-old female C57BL/6 mice. The mice treated with in situ cross-linked GO-GelMA composite hydrogel containing GO of the corresponding final mass concentration were divided into 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group according to the random number table, with 4 mice in each group. The general condition of wound was observed and the wound healing rate was calculated on 3, 7, and 14 d of treatment, the wound blood perfusion was detected by laser Doppler flowmetry on 3, 7, and 14 d of treatment and the mean perfusion unit (MPU) ratio was calculated, and the wound vascularization on 7 d of treatment was observed after hematoxylin-eosin staining and the vascular density was calculated (n=3). The wound tissue of mice in 0 μg/mL GO composite hydrogel group and 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was collected to observe the relationship between the distribution of GO and neovascularization by hematoxylin-eosin staining (n=3) and the expression of VEGF by immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey's method. Results: GO had a multilayered lamellar structure with the width of about 20 μm and the length of about 50 μm. The absorbance value of HSFs in 10.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group after 48 h of culture (q=7.64, P<0.01). At 24 h after scratching, the migration rates of HSFs were similar in the four groups (P>0.05); at 36 h after scratching, the migration rate of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.48, 10.81, and 10.20, respectively, P<0.01). At 12 h after scratching, the migration rate of HUVECs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.11, 8.99, and 14.92, respectively, P<0.01), and the migration rate of HUVECs in 5.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group and 1.0 μg/mL GO group (with q values of 7.81 and 5.33, respectively, P<0.05 or P<0.01 ). At 4 and 6 h of culture, the VEGF expressions of HSFs in the four groups were similar (P>0.05); at 8 h of culture, the VEGF expression of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group and 5.0 μg/mL GO group (with q values of 4.75 and 4.48, respectively, P<0.05). The GO-GelMA composite hydrogels in the four groups were all red liquid before cross-linking, which turned to light yellow gel after cross-linking, with no significant difference in fluidity. The GO in the GO-GelMA composite hydrogel of 0 μg/mL GO composite hydrogel group had no release of GO at all time points; the GO in the GO-GelMA composite hydrogels of the other 3 groups was partially released on 3 d of soaking, and all the GO was released on 7 d of soaking. From 3 to 14 d of treatment, the wounds of mice in the 4 groups were covered with hydrogel dressings, kept moist, and gradually healed. On 3, 7, and 14 d of treatment, the wound healing rates of mice in the four groups were similar (P>0.05). On 3 d of treatment, the MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 10.70, 11.83, and 10.65, respectively, P<0.05 or P<0.01). On 7 and 14 d of treatment, the MPU ratios of wound of mice in the four groups were similar (P>0.05). The MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was significantly lower than that on 3 d of treatment (q=14.38, P<0.05), and that on 14 d of treatment was significantly lower than that on 7 d of treatment (q=27.78, P<0.01). On 7 d of treatment, the neovascular density of wound of mice on 7 d of treatment was 120.7±4.1 per 200 times of visual field, which was significantly higher than 61.7±1.3, 77.7±10.2, and 99.0±7.9 per 200 times of visual field in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 12.88, 7.79, and 6.70, respectively, P<0.01), and the neovascular density of wound of mice in 1.0 μg/mL GO composite hydrogel group and 5.0 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group (with q values of 5.10 and 6.19, respectively, P<0.05). On 7 d of treatment, cluster of new blood vessels in wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly more than that in 0 μg/mL GO composite hydrogel group, and the new blood vessels were clustered near the GO; a large amount of VEGF was expressed in wound of mice in 0.1 μg/mL GO composite hydrogel group in the distribution area of GO and new blood vessels. Conclusions: GO with mass concentration lower than 10.0 μg/mL had no adverse effect on proliferation activity of HSFs, and GO of 0.1 μg/mL can promote the migration of HSFs and HUVECs, and can promote the secretion of VEGF in HSFs. In situ photopolymerized of GO-GelMA composite hydrogel dressing can promote the wound neovascularization of full-thickness skin defect in mice and increase wound blood perfusion in the early stage, with GO showing an enrichment effect on angiogenesis, and the mechanism may be related to the role of GO in promoting the secretion of VEGF by wound cells.
Anhydrides ; Animals ; Endothelial Cells ; Eosine Yellowish-(YS) ; Female ; Gelatin/pharmacology* ; Graphite ; Hematoxylin ; Humans ; Hydrogels/pharmacology* ; Methacrylates ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; Skin Abnormalities ; Vascular Endothelial Growth Factor A

The main objective of this study was to observe the adhesion, proliferation and differentiation of mouse osteblast-like MC3T3-E1 cells cultured on maleic anhydride-modified poly(D,L-lactic acid) (MPLA) and poly(D,L-lactic acid) (PDLLA) polymers, and to evaluate the cytocompatibility of MPLA polymer. The effects of MPLA and PDLLA polymers on the morphology, adhesion, proliferation, the content of total cellular protein, alkaline phosphatase (ALP) activity and the content of Ca of MC3T3-E1 cells were explored. These results indicated that MC3T3-E1 cells on MPLA polymer adhered and spread more fully. On MPLA polymer, the proliferation, total protein content, ALP activity, Ca content of the cells were significantly higher than those of the cells on PDLLA polymer (P < 0.01). It was concluded that MPLA polymer could promote the adhesion, spreading, proliferation and the synthesis of protein of osteoblasts, and also induced the differentiation and mineralization of osteoblasts, suggesting that MPLA polymer might have the better cytocompatibility than PDLLA.
Animals ; Biocompatible Materials ; chemistry ; pharmacology ; Cell Adhesion ; drug effects ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Embryo, Mammalian ; Lactic Acid ; chemistry ; pharmacology ; Maleic Anhydrides ; chemistry ; pharmacology ; Mice ; Osteoblasts ; cytology ; Polyesters ; Polymers ; chemistry ; pharmacology

AIMTo investigate the bio-affinities of ligustilide and butylidenephthalide to rat aortic smooth muscle cells and the inhibitory effects of them on bFGF-stimulated proliferation of rat vascular smooth muscle cell (VSMC).

METHODSVSMCs were cultured from rat aorta pectoralis and identified by an immunohistochemical method. The bio-affinities between solute (ligustilide or butylidenephthalide) and cell membrane were measured by rat aortic cell membrane chromatography (CMC). The inhibitory effects of ligustilide and butylidenephthalide on bFGF-stimulated VSMC proliferation were evaluated by MIT colorimetric method.

RESULTSBoth ligustilide and butylidenephthalide had selective affinities to rat aortic smooth muscle cell as the same as verapamil, one of the calcium ion antagonists. They could potently inhibit the bFGF-stimulated VSMC proliferation at the concentrations of 5.5 and 11.1 micromol x L(-1), separately (P < 0.05), but had no effects on the normal VSMC growth.

CONCLUSIONBoth ligustilide and butylidenephthalide can inhibit the abnormal proliferation of VSMC induced by bFGF.

4-Butyrolactone ; analogs & derivatives ; isolation & purification ; pharmacology ; Animals ; Aorta, Thoracic ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Fibroblast Growth Factor 2 ; antagonists & inhibitors ; Ligusticum ; chemistry ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Phthalic Anhydrides ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley