Hydroxyproline Content Research Articles

Combined Silymarin and Cotrimoxazole Therapy Attenuates Pulmonary Fibrosis in Experimental Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), which mainly affects rural workers, is a systemic mycosis caused by the Paracoccidioides genus that induces pulmonary sequelae in most adult patients, causing serious disability and impairing their quality of life. Silymarin is herbal medicine with an effective antifibrotic activity. Considering that in PCM, antifibrotic treatment is still not available in pulmonary fibrosis, we aimed to evaluate combined silymarin and cotrimoxazole (CMX) therapy via the intratracheal route in BALB/c mice infected with P. brasiliensis yeast. After 12 weeks of treatment, the lungs were collected for the determination of fungal burden, production of OH-proline, deposition of collagen fibers, pulmonary concentrations of cytokines, and expression of fibronectin, α-SMA, MMP-2, MMP-9, and TIMP-2. Spleen cell cultures were also performed. Our results showed that infected mice treated with combined silymarin/CMX showed lower deposition of collagen fibers in the lungs and lower pulmonary concentrations of hydroxyproline than the placebo groups. Decreased levels of TGF-β1 and fibronectin and high levels of MMP-2 and IFN-γ were also observed in this group of mice. Collectively, our findings indicate that the combination of antifungal treatment with silymarin has a potent antifibrotic effect associated with an immunomodulatory effect that potentializes the antifungal immune response.

Evolution of conformation and thermal properties of bovine hides collagen in the sodium sulphide solution

Conformations of secondary and super secondary structure from bovine hides collagen are very important for constructing collagen molecular space structure, and directly related to its properties. In this work, the evolutional mechanism of the collagen conformation in the environment of sodium sulphide (Na2S) solution is periodically studied by FT-IR spectroscopy, the Gaussian peak fitting of secondary derivative, and auxiliary methods such as SDS-PAGE, SEM and TG. As a result, the three polypeptide chains structure of bovine hide collagen was hardly affected by the ions of OH−, HS− and S2−. The conformation of collagen molecules such as β-turn, triple helix and β-strand were gradually evolved into unordered structure and α-helix with the sequential addition of OH−, HS− and S2−, which is because the hydrogen bond inside collagen molecular is extended or opened with the increase of the synergistic effect of pH and reducibility of HS− and S2− in the solution. The bound water content and thermal properties of the treated collagen molecules are decreased. Finally, the order of the hydrolysis ability to collagen is NaOH < NaHS < Na2S at the same condition, which was confirmed by measuring the content of hydroxyproline from their solutions after treating the collagen. This work provides very important information for understanding the evolutional mechanism of bovine hide collagen conformation in the Na2S solution, developing the alternatives of sulfide, extraction of collagen, tanning, and application of collagen in biological and material science.

Muscle contractile exercise through a belt electrode device prevents myofiber atrophy, muscle contracture, and muscular pain in immobilized rat gastrocnemius muscle.

Immobilization of skeletal muscles causes muscle atrophy, muscle contracture, and muscle pain, the mechanisms of which are related to macrophage accumulation. However, muscle contractile exercise through a belt electrode device may mitigate macrophage accumulation. We hypothesized that such exercise would be effective in preventing myofiber atrophy, muscle contracture, and muscular pain. This study tested this hypothesis in immobilized rat gastrocnemius muscle. A total of 32 rats were divided into the following control and experimental groups: immobilization (immobilized treatment only), low-frequency (LF; immobilized treatment and muscle contractile exercise with a 2 s (do) /6 s (rest) duty cycle), and high-frequency (HF; immobilized treatment and muscle contractile exercise with a 2 s (do)/2 s (rest) duty cycle). Electrical stimulation was performed at 50 Hz and 4.7 mA, and muscle contractile exercise was applied to the lower limb muscles for 15 or 20 min/session (once daily) for 2 weeks (6 times/week). After the behavioral tests, the bilateral gastrocnemius muscles were collected for analysis. The number of macrophages, the Atrogin-1 and MuRF-1 mRNA expression, and the hydroxyproline content in the HF group were lower than those in the immobilization and LF groups. The cross-sectional area (CSA) of type IIb myofibers in the superficial region, the PGC-1α mRNA expression, and the range of motion of dorsiflexion in the HF group were significantly higher than those in the immobilization and LF groups. The pressure pain thresholds in the LF and HF groups were significantly higher than that in the immobilization group, and the nerve growth factor (NGF) content in the LF and HF groups was significantly lower than that in the immobilization group. Muscle contractile exercise through the belt electrode device may be effective in preventing immobilization-induced myofiber atrophy, muscle contracture, and muscular pain in the immobilized rat gastrocnemius muscle.

An efficient strategy to recellularization of a rat aorta scaffold: an optimized decellularization, detergent removal, and Apelin-13 immobilization

BackgroundTissue engineering of native vessels is an alternative approach for patients with vascular disease who lack sufficient saphenous vein or other suitable conduits for autologous vascular graft. Moreover, the harvest of vessels prolongs the surgical procedure and it may lead to the morbidity of donor site in elder patients: therefore, it seems that the use of tissue-engineered vessels would be an attractive and less invasive substitute for autologous vascular grafts. Apelin-13 plays a pivotal role in cell proliferation, survival, and attachment; therefore, covalent attachment of apelin-13 to the acellular scaffolds might be a favorable approach for improving recellularization efficacy.MethodsIn the present study, the decellularization process was performed using various detergents. Afterward, the efficacy of decellularization procedure was evaluated using multiple approaches including assessment of DNA, hydroxyproline, and GAG content as well as Masson’s trichrome and orcein staining used for collagen and elastin determination. Subsequently, the scaffold was bioconjugated with apelin-13 using the EDC-NHS linker and acellular scaffolds were recellularized using fibroblasts, endothelial cells, and smooth muscle cells. SEM images and characterization methods were also used to evaluate the effect of apelin-13 attachment to the acellular scaffold on tissue recellularization. We also developed a novel strategy to eliminate the remnant detergents from the scaffold and increase cell viability by incubating acellular scaffolds with Bio-Beads SM-2 resin. Testometric tensile testing machine was also used for the assessment of mechanical properties and uniaxial tensile strength of decellularized and recellularized vessels compared to that of native tissues.ResultsOur results proposed 16-h perfusion of 0.25% sodium dodecyl sulfate (SDS) + 0.5% Triton X-100 combination to the vessel as an optimal decellularization protocol in terms of cell elimination as well as extracellular matrix preservation. Furthermore, the results demonstrated considerable elevation of cell adhesion and proliferation in scaffolds bioconjugated with apelin-13. The immunohistochemical (IHC) staining of CD31, α-SMA, and vimentin markers suggested placement of seeded cells in the suitable sites and considerable elevation of cell attachment within the scaffolds bioconjugated with apelin-13 compared to the non-bioconjugated, and decellularized groups. Moreover, the quantitative analysis of IHC staining of CD31, α-SMA, and vimentin markers suggested considerable elevation in the number of endothelial, smooth muscle, and fibroblast cells in the recellularized scaffolds bioconjugated with apelin-13 group (1.4% ± 0.02, 6.66% ± 0.23, and 9.87% ± 0.13%, respectively) compared to the non-bioconjugated scaffolds (0.03% ± 0.01, 0.28% ± 0.01, and 1.2% ± 0.09%, respectively) and decellularized groups (0.03% ± 0.007, 0.05% ± 0.01, and 0.13% ±0.005%, respectively). Although the maximum strain to the rupture was reduced in tissues decellularized using 0.5% SDS and CHAPS compared to that of native ones (116% ± 6.79, 139.1% ± 3.24, and 164% ± 8.54%, respectively), ultimate stress was decreased in all decellularized and recellularized groups. Besides, our results indicated that cell viability on the 1st, 3rd, and 7th day was 100.79% ± 0.7, 100.34% ± 0.08, and 111.24% ± 1.7% for the decellularized rat aorta conjugated with apelin-13, which was incubated for 48-h with Bio-Beads SM-2, and 73.37% ± 7.99, 47.6% ± 11.69, and 27.3% ± 7.89% for decellularized rat aorta scaffolds conjugated with apelin-13 and washed 48-h by PBS, respectively. These findings reveal that the incubation of the scaffold with Bio-Beads SM-2 is a novel and promising approach for increasing cell viability and growth within the scaffold.ConclusionsIn conclusion, our results provide a platform in which xenograft vessels are decellularized properly in a short time, and the recellularization process is significantly improved after the bioconjugation of the acellular scaffold with apelin-13 in terms of cell adhesion and viability within the scaffold.Graphical

Lack of ZNF365 Drives Senescence and Exacerbates Experimental Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant activation of the alveolar epithelium, the expansion of the fibroblast population, and the accumulation of extracellular matrix. Global gene expression of human lung fibroblasts stimulated with TGFβ-1, a strong fibrotic mediator revealed the overexpression of ZNF365, a zinc finger protein implicated in cell cycle control and telomere stabilization. We evaluated the expression and localization of ZNF365 in IPF lungs and in the fibrotic response induced by bleomycin in WT and deficient mice of the orthologous gene Zfp365. In IPF, ZNF365 was overexpressed and localized in fibroblasts/myofibroblasts and alveolar epithelium. Bleomycin-induced lung fibrosis showed an upregulation of Zfp365 localized in lung epithelium and stromal cell populations. Zfp365 KO mice developed a significantly higher fibrotic response compared with WT mice by morphology and hydroxyproline content. Silencing ZNF365 in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and increased senescence markers, including Senescence Associated β Galactosidase activity, p53, p21, and the histone variant γH2AX. Our findings demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and suggest a protective role since its absence increases experimental lung fibrosis mechanistically associated with the induction of cell senescence.

Lactacystin-induced kidney fibrosis: Protection by melatonin and captopril.

Lactacystin is a specific proteasome inhibitor that blocks the hydrolysis of intracellular proteins by ubiquitin/proteasome system inhibition. The administration of lactacystin to rats induced hypertension and remodeling of the left ventricle and aorta. This study tested whether lactacystin induces structural and fibrotic rebuilding of the kidneys and whether melatonin and captopril can prevent these potential changes. Six weeks of lactacystin administration to rats increased their average systolic blood pressure (SBP). In the kidneys, lactacystin reduced glomerular density, increased the glomerular tuft area, and enhanced hydroxyproline concentrations. It also elevated the intraglomerular proportion including the amounts of collagen (Col) I and Col III. Lactacystin also raised the tubulointerstitial amounts of Col I and the sum of Col I and Col III with no effect on vascular/perivascular collagen. Six weeks of captopril treatment reduced SBP, while melatonin had no effect. Both melatonin and captopril increased glomerular density, reduced the glomerular tuft area, and lowered the hydroxyproline concentration in the kidneys. Both drugs reduced the proportion and total amounts of intraglomerular and tubulointerstitial Col I and Col III. We conclude that chronic lactacystin treatment stimulated structural and fibrotic remodeling of the kidneys, and melatonin and captopril partly prevented these alterations. Considering the effect of lactacystin on both the heart and kidneys, chronic treatment with this drug may be a prospective model of cardiorenal damage suitable for testing pharmacological drugs as protective agents.

Zerumbone-Loaded Nanostructured Lipid Carrier Gel Enhances Wound Healing in Diabetic Rats.

This study investigated the healing effects of topical application of zerumbone, a well-known anti-inflammatory compounds loaded on nanostructured lipid carrier gel (Carbopol 940) (ZER-NLCG) on excisional wounds in streptozotocin-induced diabetic rats. Diabetic rats with inflicted superficial skin wound were topically treated with ZER-NLCG, empty NLCG, and silver sulfadiazine cream (SSDC) once daily for 21 days. Wound tissue samples were analyzed for proinflammatory cytokines, namely, interleukin-6 (IL-6), interleukin-1 β (IL-1β), and tumor necrosis factor-α (TNF-α), hydroxyproline contents, catalase, superoxide dismutase activities, and lipid peroxidation level, and were subjected to histopathological analysis, respectively. Among the treated groups, ZER-NLCG was the most effective at decreasing proinflammatory cytokine level and inflammatory cell infiltration while increasing antioxidant enzyme activities, hydroxyproline content, and granulation of wound tissues of diabetic rats. ZER-NLCG is a potent formulation for the enhancement of wound healing in diabetic rats through its anti-inflammatory, antioxidant, and tissue repair activities.

Jian Pi Shen Shi formula alleviates hyperuricemia and related renal fibrosis in uricase-deficient rats via suppression of the collagen-binding pathway.

Jian Pi Shen Shi Formula (JPSSF) is a beneficial treatment for hyperuricemia and related tissue damage in the clinical setting. This study was designed to investigate its therapeutic potential and underlying mechanisms in uricase-deficient rats (Uox-/- rats). Uox-/- rats were used to assess the therapeutic potential of JPSSF on hyperuricemia. Protein extracts from renal tissues of a Uox-/- group and a JPSSF group were analyzed using tandem mass tag labeling quantitative proteomic workflow. Collagen deposition in Uox-/- rat kidneys was analyzed by Masson trichromatic staining. The gene expression associated with collagen-binding-related signaling pathways in the kidneys was further explored using quantitative polymerase chain reaction assay. The protein expressions of collagen 1a1 (col1a1), col6a1, and α-smooth muscle actin were measured by Western blotting and immunohistochemistry. JPSSF significantly decreased renal function indices and alleviated renal injuries. The action of JPSSF was manifested by down-regulation of col6a1 and interleukin-1 receptor-associated kinase-like 2, which blocked the binding sites on collagen and further prevented kidney injury. The anti-renal fibrosis effect of JPSSF was confirmed by reducing the collagen deposition and hydroxyproline concentrations. JPSSF treatment also intensely down-regulated the mRNA and protein expressions of col6a1, col1a1, and α-smooth muscle actin, which inhibited the function of the collagen-binding-related signaling pathway. Our results indicated that JPSSF notably ameliorated hyperuricemia and related renal fibrosis in Uox-/- rats through lowering uric acid and down-regulating the function of the collagen-binding pathway. This suggested that JPSSF is a potential empirical formula for treating hyperuricemia and accompanying renal fibrosis.

Eicosapentaenoic acid loaded silica nanoemulsion attenuates hepatic inflammation through the enhancement of cell membrane components

BackgroundLiver inflammation is a multistep process that is linked with cell membrane fatty acids composition. The effectiveness of eicosapentaenoic acid (EPA) undergoes an irreversible change during processing due to their unsaturated nature; so the formation of nanocarrier for EPA is crucial for improving EPA’s bioavailability and pharmacological properties.ObjectiveIn this study we aimed to evaluate the efficiency of EPA alone or loaded silica nanoemulsion on the management of hepatic inflammation induced by diethyl nitrosamine (DEN) through the enhancement of the cell membrane structure and functions.MethodsThe new formula of EPA was prepared to modify the properties of EPA. Forty-eight male Wistar albino rats were classified into: control, EPA, EPA loaded silica nanoemulsion (EPA–NE), DEN induced hepatic inflammation; DEN induced hepatic inflammation treated with EPA or EPA –NE groups. Plasma tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), liver hydroxyproline (Hyp) content, and liver oxidant and anti-oxidants were estimated. Urinary 8- hydroxyguanozine (8- OHdG) and erythrocyte membrane fatty acids fractions were estimated by High-performance liquid chromatography (HPLC). Also, histopathology studies were done to verify our hypothesis.ResultsIt was appeared that administration of EPA, in particular EPA loaded silica nanoemulsion, ameliorated the inflammatory response, increased the activity of the anti-oxidants, reduced levels of oxidants, and improved cell membrane structure compared to hepatic inflammation induced by DEN group. Histopathological examination confirmed these results.ConclusionEPA and notably EPA loaded silica nanoemulsion strongly recommended as a promising supplement in the management of hepatic inflammation.

Indicators of connective tissue biopolymers in healthy pregnant women

Objective. To detect the features of connective tissue metabolism in healthy women during pregnancy.&#x0D; Materials and methods. The study of the connective tissue components was conducted at the terms of 1516 weeks, 2024 weeks as well as before the labor. Under observation there were 52 pregnant women (main group), who underwent the study of the connective tissue components in dynamics of pregnancy and 39 patients (comparison group) the study of the connective tissue biopolymers before pregnancy. The main protein of connective tissue collagen was studied by a quantitative determination of amino acids of the free- and peptide-bound hydroxyproline. Metabolism of glycoproteins was investigated according to the concentrations of free oligo-bound and protein-bound sialic acids, oligo-bound and protein-bound fucose, fucosidose and hyaluronidase activity. Seromucoid, hexasamine and glycosaminoglycan content was analyzed.&#x0D; Results. There was established a statistically significant increase in the blood serum free and piptide-bound hydroxyproline content, the absence of changes in the blood serum glycoprotein concentration, the growth of glycosaminoglycan concentrations and fucosidase concentrations beginning from the week 20 of pregnancy, the elevation of protein-bound sialic acid concentration from the weeks 2024 of pregnancy, free and oligo-bound sialic acids from the week 37 in the blood serum.&#x0D; Conclusions. The study results indicate that among healthy women, disorganization and structural changes in the connective tissue during pregnancy are based on the changes in the anabolic and catabolic processes conditioned by improvement of the humoral control of metabolism.

Impact of different ultrasound-assisted processes for preparation of collagen hydrolysates from Asian bullfrog skin on characteristics and antioxidative properties

This study focused on impact of ultrasound-assisted process (UAP) at pre-treatment (UP) and simultaneous treatment (US) during papain hydrolysis for preparing collagen hydrolysate (CH) from Asian bullfrog skin. Ultrasonication times were varied (10, 20, 30 min), and CH prepared using papain hydrolysis without UAP was used as control. Different UAPs provided CH with various hydroxyproline contents, α-amino group contents, surface hydrophobicities, and antioxidative activities. UP at 20 min (UP-20) and US at 30 min (US-30) provided highly antioxidative CHs, which were selected for further studies on their Oxygen reactive absorbance capacity (ORAC) and molecular characteristics. CHs from UP-20 and US-30 had higher ORAC than that of control group (p ≤ 0.05). Slight difference in amino acid composition was detected between samples. Based on these results, molecular characteristic styles, molecular weight profile, antioxidative peptide content, and secondary structure of each sample were obtained. These results indicate that UP and US used varied enzymatic hydrolysis patterns and modified molecular conformation of CH, resulting in enhanced antioxidative activity. Therefore, different UAPs as UP and US could be effectively used in preparation of CH using papain hydrolysis from Asian bullfrog skin, which could improve production process efficiency by enhancing their bioactivity, particularly antioxidative activity.

Intramuscular connective tissue content and mechanical properties: Influence of aging and physical activity in mice.

Aging is accompanied by morphological and mechanical changes to the intramuscular connective tissue (IMCT) of skeletal muscles, but whether physical exercise can influence these changes is debated. We investigated the effects of aging and exercise with high or low resistance on composition and mechanical properties of the IMCT, including direct measurements on isolated IMCT which has rarely been reported. Middle-aged (11months, n=24) and old (22months, n=18) C57BL/6 mice completed either high (HR) or low (LR) resistance voluntary wheel running or were sedentary (SED) for 10weeks. Passive mechanical properties of the intact soleus and plantaris muscles and the isolated IMCT of the plantaris muscle were measured in vitro. IMCT thickness was measured on picrosirius red stained cross sections of the gastrocnemius and soleus muscle and for the gastrocnemius hydroxyproline content was quantified biochemically and advanced glycation end-products (AGEs) estimated by fluorometry. Mechanical stiffness, IMCT content and total AGEs were all elevated with aging in agreement with previous findings but were largely unaffected by training. Conclusion: IMCT accumulated with aging with a proportional increase in mechanical stiffness, but even the relatively high exercise volume achieved with voluntary wheel-running with or without resistance did not significantly influence these changes.

Динаміка показників білкового обміну ротової рідини після комплексного лікування генералізованого пародонтиту у осіб молодого віку на тлі первинного гіпотиреозу

The purpose of the study was to evaluate the effectiveness of complex treatment of generalized periodontitis in young people with primary hypothyroidism based on protein metabolism indicators of the oral fluid. Materials and methods. Complex treatment of chronic generalized periodontitis was carried out in 110 patients aged 25–44, who were divided into treatment groups: the main group – 55 patients with hypothyroidism, in whom the developed treatment and prevention complex was used; and the comparison group, which included 55 patients with hypothyroidism, where the treatment was carried out according to the “Protocols of the Ministry of Health of Ukraine for the provision of medical care” in the specialty “Therapeutic dentistry”. The activity of elastase, collagenase, and the content of hydroxyproline in the oral fluid of individuals in the treatment groups were evaluated. Results and discussion. 1 month after comprehensive treatment of generalized periodontitis, the activity of the proteolytic destructive enzyme elastase in the oral fluid of the main group significantly decreased by 3.3 times: from 1.14 ± 0.12 μkat/l to 0.35 ± 0.06 μkat/l, p&lt;0.01. Collagenase activity also decreased by 1.7 times from the digital value of 0.50 ± 0.06 mmol/l to 0.29 ± 0.04 mmol/l, p&lt;0.01. In the current period of observation, in the main group, on the other hand, an increase by 1.4 times in the level of protein-bound hydroxyproline in the oral fluid was observed: from 3.03 ± 0.22 μg/ml to 4.25 ± 0.54 μg/ml, p&lt;0.01. In the main group, 6 months after using the proposed therapeutic and preventive scheme, the stabilization of the indicated indicators was confirmed: the activity of elastase in the oral fluid was 0.36 ± 0.06 μkat/l, the activity of collagenase was equal to 0.32 ± 0.05 mmol/l, the level of protein-bound hydroxyproline in oral fluid was within laboratory norms – 4.16 ± 0.52 μg/ml, p&lt;0.01. 12 months after the treatment of generalized periodontitis in young people with hypothyroidism, who were treated according to the developed scheme, all indicators of protein metabolism of the oral fluid were within the reference range. In patients of the comparison group, positive dynamics of indicators of protein metabolism of oral fluid were observed only in the near term of observation, in the distant terms, the indicated indicators were at the lower limit of the margin and were significantly lower than the initial values before treatment. Conclusion. In young people with hypothyroidism, after comprehensive treatment of generalized periodontitis according to the developed scheme, a decrease in the activity of the destructive enzymes elastase and collagenase and an increase in the concentration of protein-bound hydroxyproline in the oral fluid were observed, which indicates the predominance of the synthetic phase over the catabolic phase in collagen metabolism

The Effect of Genistein on Anastomotic Healing in Intestinal Ischemia/Reperfusion Injury

IntroductionGenistein is a natural isoflavonoid and has several pharmacological effects, such as antioxidant, antitumor activity, and improvement of glucose metabolism. The safety of intestinal anastomosis after ischemia–reperfusion (I/R) injury is a critical issue for surgeons. This experimental study aimed to investigate the effects of genistein on anastomotic healing after intestinal I/R injury. MethodsA total of 36 male Wistar Albino rats were divided into four groups: control, I/R, genistein, and genistein + I/R. The control group received segmental ileal resection and ileoileal anastomosis. The I/R group received resection + anastomosis after intestinal I/R. The genistein group was administered subcutaneous injection of 1 mg/kg genistein 12 h and 1 h before the procedure and received ileal resection + anastomosis. The genistein + I/R group received I/R + ileal resection + anastomosis after genistein injection. Anastomotic bursting pressure, hydroxyproline, superoxide dismutase, and glutathione peroxidase levels and histopathological wound healing scores of all rats were measured on postoperative day 5. ResultsThe anastomotic bursting pressure was significantly higher in the genistein and genistein + I/R groups (P < 0.001). Genistein increased the hydroxyproline concentration and the superoxide dismutase and glutathione peroxidase levels in the intestinal anastomosis (P < 0.001). In histopathological assessment, the mean wound healing score was significantly higher in the genistein group than in the other groups (P < 0.001). ConclusionsGenistein, with its anti-inflammatory and antioxidant properties, shows protective effects against increased oxidative stress after intestinal I/R injury and contributes positively to intestinal anastomotic healing.

Efficacy of Human-Recombinant Epidermal Growth Factor Combined with Povidone-Iodine for Pressure Ulcers and Its Influence on Inflammatory Cytokines.

To determine the clinical efficacy of recombinant human epidermal growth factor (rh-EGF) combined with povidone-iodine (PVI) on patients with pressure ulcers (PUs). One hundred and five PU patients treated between January 2018 and January 2021 were enrolled and retrospectively analyzed. Of them, 50 patients who received conventional treatment were assigned to the control group (Con group), while 55 patients treated with rh-EGF combined with PVI were assigned to the observation group (Obs group). The two groups were compared in clinical efficacy, PU alleviation (total area reduction rate, total depth reduction rate, and total volume reduction rate), healing time, pain degree (Visual Analog Scale [VAS] score), inflammatory indexes (interleukin-8 [IL-8], tumor necrosis factor-α [TNF-α], and hypersensitive C reactive protein [hs-CRP]), and hydroxyproline content in the wound. The Obs group yielded a higher total effective rate than the Con group (P < 0.05). The Obs group also experienced statistically shorter healing time and milder pain, with better PU alleviation and lower levels of inflammation indexes compared with the Con group (all P < 0.05). In addition, a higher hydroxyproline content in the wound was found in the Obs group. All in all, rh-EGF combined with PVI has a definite curative effect on patients with PUs. It can promote PU alleviation and hydroxyproline secretion in the wound and inhibit pain and inflammatory reactions, which is worthy of clinical promotion.

Gelatin extracted from jundiá skin (Rhamdia quelen): An alternative to the discarded by-product

The production of gelatin from by-products of the fishing industry values ​​the discarded raw material and serves a part of the population that does not consume products originating from mammals. Therefore, the objective of the research was to use the jundiá skin (Rhamdia quelen) (JS) to obtain gelatin (GJS) and characterize this product, not yet studied until the present moment. Thus, the extraction process showed a yield of 7.3 % for JS and 18.2 % for GJS (in wet weight). Both JS and GJS presented, in their composition, high concentration of protein (26.3 and 88.1 %), low levels of fixed mineral residue (1.0 and 1.9 %), lipids (1.7 and 1.5 %) and hydroxyproline content (1.5 and 7.2 %), respectively. The GJS dispersion had a pH value of 4.7 and the color analysis indicated a snow effect with a white appearance. Fourier transform infrared spectroscopy (FTIR) showed amide bands commonly found in gelatin, gel electrophoresis (SDS-PAGE) showed high molecular weight bands, differential scanning calorimetry (DSC) revealed a denaturation temperature of 69.4 °C and scanning electron microscopy (SEM) showed a compact and non-porous structure. The emulsifying property was high when subjected to a temperature of 80 °C for 30 min, while the foaming capacity was significant at a concentration of 1 %. The highest dispersivity was observed at pH 2.0 and, in this condition, the viscosity was higher than that of other gelatin sources (25.5 cP). In view of the above, attention is drawn to the use of JS as a raw material for obtaining gelatin and for the various possibilities of application.

In Vitro Evaluation of the Permeability of Different Resorbable Xenogeneic Membranes after Collagenolytic Degradation.

In this in vitro study, we compare the penetration of cells through different resorbable collagen membranes, which were collagenolytically degraded over different time periods. Three different resorbable collagen membranes were evaluated, including two non-cross-linked (NCL) membranes—namely, a porcine (NCL-P) and an equine (NCL-E) membrane—and an enzymatically cross-linked porcine (ECL-B) membrane. A special two-chamber model was fabricated, allowing for the placement of separating membranes, and a non-porous polyester membrane was used as a negative control (C), in order to verify the impermeability of the experimental chamber device. Round membrane samples with a diameter of 16 mm were fabricated. Eighteen membranes of each type were punched and placed on polyethylene nets as carriers. The membranes were then biodegraded—each on its carrier—in 12-well polystyrene plates: three samples of each membrane type were degraded for 1.5, 3, 6, or 12 h in 2 mL of a buffered collagenase solution, at 37 °C. For control purposes, three samples of each membrane type were not degraded, but only immersed in buffer solution for 1.5, 3, 6, or 12 h, at 37 °C. Another three samples of each type of membrane were degraded until complete dissolution, in order to determine the full hydroxyproline content for comparison. Liquid-preserved boar semen (containing at least 120 million sperm cells per milliliter) was used to test the cell occlusivity of the degraded membranes. At baseline and initial degradation, all tested membranes were tight, and no penetration was observed with up to 30 min of incubation time (results not shown). After 1.5 h, cells were partially capable of penetrating the NCL-E membrane only. One sample showed leakage, with a sperm volume of 1.7 million cells/mL over all samples. No penetration occurred in the test, NCL-P, and ECL-B groups. After a degradation time of 3 h, the NCL-P and ECL-B membranes remained occlusive to cells. All the membranes and measurements indicated leakage in the NCL-E group. After 6 h, four NCL-P measurements showed the first signs of cell penetration, as boar spermatozoa were detectable in the lower chamber (64 million cells/mL). The ECL-B membranes remained completely cell occlusive. After 12 h, four NCL-P measurements were cell penetration positive (14.6 million cells/mL), while the ECL-B group remained tight and showed no cell penetration. As the findings of our study are well in accordance with the results of several previous animal studies, it can be concluded that the surrogate model is capable of performing rapid and cheap screening of cell occlusivity for different collagen membranes in a very standardized manner. In particular, claims of long degradation resistance can be easily proven and compared. As the boar spermatozoa used in the present report had a size of 9 × 5 μm, smaller bacteria are probably also able to penetrate the leaking membranes; in this regard, our proposed study set-up may provide valuable information, although it must be acknowledged that sperm cells show active mobility and do not only translocate by growth.

The value of undenatured collagen for the normalization of the function of the cartilaginous tissue of the joints

Among the proteins of the human body, collagen accounts for at least 25–45% collagen; it is an essential structural component of skeletal tissues, connective tissue of internal organs (heart, intestines, lungs, liver, kidneys), as well as blood vessels. The variety of types of collagen is due to their role and function. The article presents information about the structure and synthesis of collagen, a high concentration of glycine, proline and hydroxyproline in the alpha chains of the collagen helix. Attention is drawn to the fact that cross-links and orientation of collagen fibers in the direction of the vector of external forces and loads provide protection to tissues and organs. The clinical significance of type II collagen is considered on the example of the cartilaginous tissue of the joints and tendons. It is emphasized that an imbalance in the structure of nutrition, genetic mutations, dysfunction of the endocrine and immune systems, especially in old age, are associated with the occurrence of one of the most common joint diseases – osteoarthritis (ОА). It has been established that the degradation or reduction of type II collagen in the cartilage matrix is accompanied by the progression of this disease. Due to the increasing prevalence of OA, signs of metabolic disorders and post-traumatic joint injuries, there is a growing interest in non-pharmacological and pharmacological interventions for the prevention and treatment of osteoarthritis. In recent years, convincing evidence has emerged of the successful use of drugs (biologically active food supplements) of collagen in osteoarthritis. The article presents the results of experimental and clinical studies, meta-analysis and systematic review, confirming the possibility of using these drugs (products) as part of the complex treatment of OA. The possibility of using compositions based on undenatured (native) collagen type II, with the inclusion of ascorbic acid, vitamin D, methylsulfonylmethane and boswellic acids promotes the synergy of these substances, slows down the rate of cartilage destruction, reduces the manifestation of pain and inflammation in the joints, improves functional joint and spinal conditions, promotes the synthesis of endogenous collagen.

Downregulation of miR-20b-5p Contributes to the Progression of Liver Fibrosis via the STAT3 Signaling Pathway In Vivo and In Vitro.

Activated hepatic stellate cells (HSCs) are primarily involved in liver fibrosis and portal hypertension (PHT). We aimed to investigate the effect of miR-20b-5p on HSCs, liver fibrosis, and PHT. MiR-20b-5p expression in HSCs and in mouse liver fibrosis was determined by qPCR. Further, the effects of miR-20b-5p mimic on HSCs migration, proliferation, and apoptosis were investigated in vitro. A dual-luciferase reporter assay was performed to confirm the direct interaction between miR-20b-5p and STAT3. In vivo, mouse liver fibrosis was established by common bile duct ligation and intervened by agomiR-20b-5p. Sirius red staining and hydroxyproline content were used to evaluate collagen deposition. The α-SMA expression in the liver was detected by IHC and Western blotting. The STAT3 signaling pathway and its downregulated cytokines as well as portal pressure and angiogenesis were explored. MiR-20b-5p was significantly downregulated during HSCs activation and in mouse liver fibrosis. The functional analyses demonstrated that miR-20b-5p inhibited cell proliferation, activation, and promoted apoptosis in HSCs in vitro. Moreover, miR-20b-5p regulated STAT3 expression by binding to the 3'UTR of its miRNA directly. Overexpression of miR-20b-5p facilitated HSC activation and proliferation by inhibiting the STAT3 signaling pathway. MiR-20b-5p overexpression suppressed the STAT3 and its downstream cytokines and ameliorated liver fibrosis in mice. The intra- and inter-hepatic angiogenesis were also effectively inhibited. The inhibition of liver fibrosis and neoangiogenesis contributed to the decrease of portal pressure. MiR-20b-5p plays an important role in the fibrosis and angiogenesis of liver fibrosis by targeting the STAT3 signaling pathway.

TNF-α inhibitor ameliorates immune-related arthritis and pneumonitis in humanized mice

ObjectivesThis study aimed at establishing a mouse model of immune-related adverse in humanized BALB/c-hPD1/hCTLA4 mice to investigate their potential pathogenesis and explore therapeutic targets for immune-related arthritis and pneumonitis.MethodsHumanized BALB/c-hPD1/hCTLA4 mice were injected with vehicle or collagen-specific antibodies (CA) and immune checkpoint inhibitors (ICI, ipilimumab, anti-human CTLA-4; and nivolumab, anti-human PD-1), and some mice were treated with anti-TNF-α antibody, leading to the control, collagen antibody-induced arthritis (CAIA), CAIA+ICI and treatment groups. The severity of clinical arthritis and pneumonitis in mice was monitored longitudinally and the pathological changes in the joints and lungs were histologically analyzed and the contents of lung hydroxyproline were measured. The frequency of different subsets of T cells was analyzed by flow cytometry and multiplex immunofluorescency.ResultsCompared with the control, the ICI group of mice developed the delayed onset of moderate degrees of arthritis while the CAIA+ICI group of mice exhibited the early onset of severe arthritis. Treatment with ICI caused severe pneumonitis, especially in the mice with CA. Flow cytometry analysis indicated a significantly higher frequency of splenic TNF-α+CD4+ and TNF-α+CD8+ T cells, but not other subsets of T cells tested, in the CAIA+ICI group of mice, relative to that in other groups of mice. Treatment with anti-TNF-α significantly mitigated the severity of arthritis and pneumonitis as well as deposition of collagen in lung of mice. The treatment also decreased the frequency of TNF-α+CD4+ and TNF-α+CD8+ T cells as well as effector memory T cells in the periphery lymph orangs and lungs of mice.ConclusionsWe successfully established a humanized mouse model of ICI-related severe arthritis and pneumonitis with a higher frequency of TNF-α+ T cells, which were significantly mitigated by anti-TNF-α treatment. Conceptually, ICI treatment can induce multiple autoimmune-like diseases in autoimmune-prone individuals and TNF-α+ T cells may be therapeutic targets for intervention of immune-related arthritis and pneumonitis.