Hydroxyproline Content Research Articles

Production of Partially Purified Collagenase from Bacillus licheniformis and It’s Use to Tenderize Aged Buffalo Meat

This study aimed to isolate and identify the Bacillus licheniformis bacteria that produces the enzyme collagenase. Due to the selective nature of this enzyme in breaking down and degrading collagen, it was used to tenderize aged buffalo meat. The bacterial isolates were screened using a selective medium and then grown on collagen-agar medium to identify enzyme-producing isolates. Thirteen enzyme-producing isolates were obtained based on the formation of a transparent corona around the colony. Biochemical assessments of the bacterial isolates were carried out using the ABIS online program. The isolates BL1–BL9, BL12 and BL13 were found to be related to B. licheniformis with a percentage match of 98%, while BL10 and BL11 were related to Bacillus pumilus and Bacillus subtilis with a percentage match of 95% and 95.3%, respectively. The enzyme activity of the isolates varied, with the highest activity reaching 200.71 units.ml-1 for isolate BL12. The enzyme of this isolate was chosen to complete the other tests. The optimal time required to obtain the highest enzymatic activity was found to be 20 h, which amounted to 210.25 units.ml-1, with a specific activity of 135.64 units.ml-1. The enzyme was partially purified using ammonium sulphate at a saturation rate of 20–70% as it showed an enzymatic activity and specificity of 355.81 units.ml-1 and a specific activity of 423.58 units.ml-1. Partially purified collagenase was used to tenderize aged buffalo meat. The results showed that treating aged buffalo meat with the enzyme solution for different immersion durations improved the pH, water-holding capacity (WHC) and cooking yield, with an increase in the concentration of hydroxyproline and a significant percentage of soluble collagen; the treated samples showed the best properties with a 60-min immersion duration.

Preparation and evaluation of sustained release pirfenidone-loaded microsphere dry powder inhalation for treatment of idiopathic pulmonary fibrosis

Pirfenidone (PFND) is a recommended oral drug used to treat idiopathic pulmonary fibrosis, but have low bioavailability and high hepatotoxicity. The study, therefore, seeks to improve the therapeutic activities of the drug via increased bioavailability and reduced associated side effects by developing a novel drug delivery system.The electrostatic spray technology was used to prepare a sustained release pirfenidone-loaded microsphere dry powder inhalation with PEG-modified chitosan (PFND-mPEG-CS-MS). The entrapment efficiency, drug loading, and in vitro cumulative drug release rate (at 24 h and with a sustained release effect) of PFND-mPEG-CS-MS were 77.35±3.01%, 11.45±0.64%, and 90.4%, respectively. The Carr's index of PFND-mPEG-CS-MS powder was 17.074±2.163% with a theoretical mass median aerodynamic diameter (MMADt) of 0.99±0.07 μm, and a moisture absorption weight gain rate (Rw) of 4.61±0.72%. The emptying rate, pulmonary deposition rate (fine particle fraction) and actual mass median aerodynamic diameter (MMADa) were 90%∼95%, 48.72±7.04% and 3.10±0.16 μm, respectively. MTT bioassay showed that mPEG-CS-MS (200 μg/mL) had good biocompatibility (RGR = 90.25%) and PFND-mPEG-CS-MS (200 μg/mL) had significant inhibitory activity (RGR = 49.82%) on fibroblast growth. The pharmacokinetic data revealed that the t1/2 (5.02 h) and MRT (10.66 h) of PFND-mPEG-CS-MS were prolonged compared with the free PFND (t1/2, 1.67 h; MRT, 2.71 h). The pharmacodynamic results also showed that the formulated-drug group had slight pathological changes, lower lung hydroxyproline content, and reduced hepatotoxicity compared with the free-drug group. The PFND-mPEG-CS-MS further significantly down-regulated TGF-β cytokines, Collagen I, and α-SMA protein expression levels compared with the free drug. The findings indicated that the PFND-mPEG-CS-MS had a good sustained release effect, enhanced bioavailability, decreased toxicity, and increased anti-fibrotic activities.

1602-P: Galectin-3 Inhibition Protects ApoE Knockout Mice against Western Diet–Induced Nonalcoholic Steatohepatitis and Glucose Intolerance

Nonalcoholic Fatty Liver Disease represents a broad spectrum of histological abnormalities ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which may progress to cirrhosis or hepatocellular carcinoma. NASH is characterized by steatosis, hepatic inflammation, hepatocellular ballooning, and fibrosis. Therefore, the aim of this work was to evaluate the effect of modified citrus pectin (MCP), inhibitor of galectin-3, in ApoE KO mice fed a western diet (WD). ApoE KO mice were divided into 3 groups: standard diet-fed (SD) group, WD-fed group, and WD-fed + MCP-treated group (WD+MCP). WD-fed mice displayed increased hepatic triacylglycerides (TAG-290%), and plasma ALT (115%) and AST (130%) compared with SD group. MCP treatment decreased hepatic TAG (30%) in WD-fed mice, plasma ALT and AST were reverted. WD-fed mice also displayed increased hepatic collagen deposition (18%), and MCP treatment inhibited this increase. These results were similar with hydroxyproline concentration in liver. WD-fed mice showed an impaired glucose tolerance compared with SD-fed mice, while MCP treatment improved glucose tolerance in WD-fed mice. Also, WD-fed mice presented increased fasting insulin (140%) compared with SD-fed mice, and MCP treatment reverted this increase by 100%. Gene expression of F4/80 was increased by 76% in the WD group compared with SD group, however had not significant difference between WD+MCP and SD group. Gene expression of matrix metalloproteinase 2 was increased by 5-fold in WD group compared with SD group, while this increase was inhibited with MCP treatment. Also, it was observed an increase in JNK phosphorylation by 3-fold in WD group, and MCP treatment inhibited this increase. Taken together, we confirm WD-fed ApoE KO mice as a suitable experimental model of NASH, and the inhibition of galectin-3 by the treatment with MCP protects ApoE KO mice against the development of NASH and glucose intolerance. Disclosure L. Araujo: None. F. Sucupira: None. C.C.B. Dias: None. A. Amaral: None. J. Camporez: None. Funding São Paulo Research Foundation (FAPESP)

Icariside Ⅱ attenuates bleomycin-induced pulmonary fibrosis by modulating macrophage polarization

Ethnopharmacological relevanceNumerous studies have provided evidence supporting the significant roles of icariin, in the prevention of multiple chronic diseases like diabetes, liver fibrosis, cardiac fibrosis, renal fibrosis, and pulmonary fibrosis. In particular, Icariside II (ISE II), a prominent flavonoid glycoside derived from Epimedium brevicornum Maxim, the principal metabolite of icariin, has demonstrated noteworthy anti-inflammatory and anti-oxidant properties, along with its ability to protect against lung remodeling. However, the research exploring ISE Ⅱ's application in treating pulmonary fibrosis remains limited. Aim of the studyThe aim of this study was to assess the therapeutic efficacy of ISE II in models of pulmonary fibrosis, while also investigating its potential mechanisms of action in cell signaling pathways. Materials and methodsAn in vitro model of pulmonary fibrosis was established by treating NIH-3T3 cells with transforming growth factor-β1 (TGF-β1). Western blot, RT-qPCR, and scratch test were performed to assess the effect of ISE Ⅱ. In addition, a murine model of pulmonary fibrosis was induced by intratracheal instillation of bleomycin, and the therapeutic effect of ISE Ⅱ was tested by orally administering ISE Ⅱ at a dose of 10 mg/kg. Three weeks later, lung function, micro-CT, hydroxyproline content, pathological staining, and cytokines detection of BALF or serum were used to assess the anti-fibrosis effects of ISE Ⅱ. Next, immunofluorescence staining, flow cytometry, and in vivo transcriptomics were used to investigate the underlying mechanisms of action. ResultsOur data revealed a significant inhibitory effect of ISE Ⅱ on the upregulation of α-smooth muscle actin (α-SMA) and collagen production induced by TGF-β1 in fibroblasts. Meanwhile, ISE Ⅱ exerted a therapeutic effect against bleomycin-induced pulmonary fibrosis in mice by improving lung function, decreasing collagen deposition, and reducing the expression of interleukin (IL)-1β, tumor necrosis factor α (TNF-α), TGF-β1 and platelet-derived growth factor (PDGF) in serum and bronchoalveolar lavage fluid (BALF). Additionally, ISE Ⅱ treatment effectively attenuated the infiltration of M2 macrophages, concurrently downregulating the expression level of M2 marker genes, such as CD206, arginase-1(Arg-1), and Chitinase-Like Protein 3 (YM-1). Importantly, we observed a statistically significant reduction in the M2 phenotype of interstitial macrophages (IMs). However, the impact of ISE Ⅱ on the M2 polarization of alveolar macrophages (AMs) did not reach statistical significance. Lastly, transcriptome sequencing results suggested that the anti-pulmonary fibrosis effects of ISE Ⅱ may be mediated by the suppression of the WNT/β-catenin signaling pathway, which modulated M2 polarization in macrophages and contributed to the amelioration of pulmonary fibrosis. By immunohistochemical analysis, it was verified that ISE Ⅱ treatment dramatically inhibited the activation of β-catenin in fibrosis murine. ConclusionOur findings indicated that ISE Ⅱ exerted anti-fibrotic effects by inhibiting pro-fibrotic macrophage polarization. The underlying mechanism of action might be mediated by modulating the WNT/β-catenin signaling pathway to inhibit the M2 program in IMs.

Effects of supplementing milk replacer with essential amino acids on blood metabolites, immune response, and nitrogen metabolism of Holstein calves exposed to an endotoxin

This study evaluated the effects of supplementing calf milk replacer with essential AA on immune responses, blood metabolites, and nitrogen metabolism of 32 Holstein bull calves [28 d of age, 44 ± 0.8 kg of body weight (BW)] exposed to lipopolysaccharide (LPS). Calves were bottle-fed a commercial milk replacer (20% crude protein and 20% fat, dry matter basis) twice daily along with a calf starter (19% crude protein, dry matter basis) for 45 d. The experiment was a randomized complete block design and treatments were a 2 × 2 factorial arrangement. Treatments were milk replacer (fed twice daily at 0.5 kg/d of powder) supplemented with or without 10 essential AA (+AA vs. -AA), and subcutaneous injection of sterile saline with or without LPS (+LPS vs. -LPS) at 3 h after the morning feeding on d 15 (4 µg LPS per kg of BW) and 17 (2 µg LPS per kg of BW). Calves also received a 2-mL subcutaneous injection of ovalbumin (6 mg of ovalbumin/mL) on d 16 and 30. Rectal temperature and blood samples were collected on d 15 before LPS injection and at h 4, 8, 12, and 24 thereafter. From d 15 to 19, total fecal and urinary output were collected, and feed refusals were documented. Rectal temperature was greater in +LPS than -LPS calves at h 4, 8, and 12 after LPS injection. Serum cortisol was greater for +LPS than -LPS at h 4 after LPS exposure. At d 28, serum antiovalbumin IgG level was greater in +LPS +AA calves compared with +LPS -AA. Serum glucose was lower for +LPS than -LPS at h 4 and 8. Serum insulin was greater in +LPS than -LPS calves. Plasma concentrations of Thr, Gly, Asn, Ser, and hydroxyproline were lower for +LPS versus -LPS calves. Plasma concentrations of Met, Leu, Phe, His, Ile, Trp, Thr, and Orn were greater in +AA calves than -AA calves. Plasma urea N and N retention were not different among LPS and AA treatments. The lower concentrations of AA in +LPS than -LPS calves indicate higher demand for AA in immuno-compromised calves fed milk replacer. Additionally, higher concentration of ovalbumin-specific IgG level in +LPS calves supplemented with +AA compared with +LPS calves with -AA suggests that supplementing AA to immune-compromised calves might improve immune status.

A study on the delayed effect of tilapia skin collagen on skin aging for mice and its possible mechanism.

Study the possible mechanism and delayed effect of tilapia skin collagen on skin aging for mice. Kunming (KM) mice were randomly divided into the aging model group, the normal group, the positive control group (vitamin E) and the low, medium, high dose tilapia skin collagen groups (2.0, 4.0, 8.0 mg/g). The normal group was only injected with saline at the back and the neck. The other groups were injected subcutaneously with 5% D-galactose and ultraviolet light jointly to establish the aging model. After modeling, the positive control group was treated with a dose of 10% vitamin E once a day, and the low, medium, high dose tilapia skin collagen group was separately applied 2.0, 4.0, 8.0 mg/g of tilapia skin collagen for 40 days. The changes of skin tissue morphology, water content, hydroxyproline (Hyp) content, and superoxide dismutase (SOD) activity in mice were studied at the day 10, 20, 30, 40, 50. Compared with the normal group, the skin of mice in the aging model group was thinner, looser, and the skin moisture content, Hyp content, SOD activity were all decreased. For mice of the low, medium, high dose tilapia skin collagen group, the thickness of dermis increased, possessing close arrangement, and the moisture content, Hyp content, SOD activity were up-regulated significantly, which effectively alleviated the aging process of skin. The dose of tilapia skin collagen was directly proportional to the anti-aging effect. Tilapia skin collagen has an obvious effect on improving skin aging.

Effects of Sex and Hunting Season on Carcass and Meat Quality Characteristics of the Brown Hare (Lepus europaeus).

The objective of the study was to determine the effects of sex and hunting season on the carcass, meat and fat quality of hunted brown hares (Lepus europaeus). Twenty-two hares of both sexes hunted in winter (December) during two hunting seasons in accordance with the law on hunting in Lithuania were evaluated using reference methods The data were subjected to two-factor analysis of variance in the general linear (GLM) procedure. No significant differences in carcass measurements and muscularity or internal organs between the sexes of brown hares were found; however, the hunting season appeared to affect the size of hares. The biceps femoris (BF) thigh muscle of males had lower (p < 0.05) dry matter content and higher (p < 0.05) drip loss compared with females. The hunting season demonstrated an effect (p < 0.001) on protein and hydroxyproline contents in the longissimus thoracis et lumborum (LTL) and affected dry matter, protein and hydroxyproline contents (p < 0.05, p < 0.001 and p < 0.01, respectively) in BF muscles, and differences in the colour of muscles were also observed. The shear force in the Warner-Bratzler (WB) test was higher (p < 0.001 and p < 0.01, respectively) for LTL and BF muscles during the first hunting season. The hunting season did not affect the total SFA in the intramuscular fat (IMF) of all the tissues, but it affected levels of monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids in the muscles. No differences were found in the total saturated fatty acids (SFA) of both muscles between the sexes, but females demonstrated lower (p < 0.05 and p < 0.01, respectively) and more favourable n-6/n-3 PUFA ratios in the muscles and fat and a lower (p < 0.05) thrombogenic (TI) index in the LTL compared with males.

S100A4-neutralizing monoclonal antibody 6B12 counteracts the established experimental skin fibrosis induced by bleomycin.

Our previous studies have demonstrated that the Damage Associated Molecular Pattern (DAMP) protein, S100A4, is overexpressed in the involved skin and peripheral blood of patients with SSc. It is associated with skin and lung involvement, and disease activity. By contrast, lack of S100A4 prevented the development of experimental dermal fibrosis. Herein we aimed to evaluate the effect of murine anti-S100A4 mAb 6B12 in the treatment of preestablished experimental dermal fibrosis. The effects of 6B12 were assessed at therapeutic dosages in a modified bleomycin-induced dermal fibrosis mouse model by evaluating fibrotic (dermal thickness, proliferation of myofibroblasts, hydroxyproline content, phosphorylated Smad3-positive cell count) and inflammatory (leukocytes infiltrating the lesional skin, systemic levels of selected cytokines and chemokines) outcomes, and transcriptional profiling (RNA sequencing). Treatment with 7.5 mg/kg 6B12 attenuated and might even reduce pre-existing dermal fibrosis induced by bleomycin as evidenced by reduction in dermal thickness, myofibroblast count and collagen content. These antifibrotic effects were mediated by the downregulation of TGF-β/Smad signalling and partially by reducing the number of leukocytes infiltrating the lesional skin and decrease in the systemic levels of IL-1α, eotaxin, CCL2 and CCL5. Moreover, transcriptional profiling demonstrated that 7.5 mg/kg 6B12 also modulated several profibrotic and proinflammatory processes relevant to the pathogenesis of SSc. Targeting S100A4 by the 6B12 mAb demonstrated potent antifibrotic and anti-inflammatory effects on bleomycin-induced dermal fibrosis and provided further evidence for the vital role of S100A4 in the pathophysiology of SSc.

#3650 LINACLOTIDE IS PROTECTIVE AGAINST RENAL ISCHEMIA-REPERFUSION INJURY

Abstract Background and Aims Renal ischemia-reperfusion injury is associated with delayed graft function and poor long-term graft survival following transplantation. Linaclotide is a guanylate cyclase C (GC-C) receptor agonist that increases intracellular cGMP concentrations and promotes intestinal transport capacity by activating the GC-C receptor. Since CKD is a deficiency of cGMP in the kidney, increasing cGMP in the kidney by linaclotide would suppress fibrosis, protecting the kidney. Method An AKI rat model of unilateral nephrectomy plus contralateral ischaemia (30 min) and impaired reperfusion (up to 14 days) was used. Linaclotide was administered before and after surgery for 2 weeks, and blood and renal tissue samples were collected to determine its effect on renal function and whether it was protective against the progression of CKD. Results In the I/R injury+ group, creatinine levels were lower in the linaclotide-treated group than in the non-treated group. Sirius red staining showed significant improvement in fibrosis. The hydroxyproline content of kidneys was determined by the basic hydroxyproline method. RNA seq showed that genes down-regulated in I/R injury+ were the oxidative stress markers SOD3 and Gpx1. The expression of genes down-regulated in I/R injury+ was up-regulated to a degree similar to that in I/R injury- by treatment with linaclotide. These data suggest that linaclotide may ameliorate renal fibrosis by suppressing renal dysfunction through its antioxidant effects. Conclusion The antioxidant effect of linaclotide was found to suppress the decrease in renal function caused by I/R. We would like to apply this finding to human renal transplantation in the future to prevent renal function deterioration due to I/R.

Elevated retinal fibrosis in experimental myopia is involved in the activation of the PI3K/AKT/ERK signaling pathway

ObjectiveThis study aimed to investigate the regulatory role of the PI3K/AKT/ERK signaling pathway in retinal fibrosis in −6.0 diopter (D) lens-induced myopic (LIM) guinea pigs. MethodsBiological measurements of eye tissues were performed on guinea pigs to obtain their refraction, axial length, retinal thickness, physiological function, and fundus retinal status. In addition, Masson staining and immunohistochemical (IHC) assay were further done to explore the changes in retinal morphology after myopic induction. Meanwhile, hydroxyproline (HYP) content was measured to evaluate the degree of retinal fibrosis. Moreover, the levels of the PI3K/AKT/ERK signaling pathway and fibrosis-related molecules in retinal tissues including matrix metalloproteinase 2(MMP2), collagen type I (Collagen I), and α-smooth muscle actin (α-SMA) were detected by real-time quantitative PCR (qPCR) and Western blot. ResultsThe LIM guinea pigs showed a significant myopic shift in refractive error and an increase in axial length compared with those of the normal control (NC) group. Masson staining, hydroxyproline content determination, and IHC showed an increase in retinal fibrosis. After myopic induction, qPCR and western blot analyses showed that phosphatidylinositol-3-kinase catalytic subunit α (PIK3CA), protein kinase B (AKT), extracellular regulated protein kinase 1/2 (ERK1/2), MMP2, Collagen I, and α-SMA were consistently elevated in the LIM group than those in the NC group. Conclusion: The PI3K/AKT/ERK signaling pathway was activated in the retinal tissues of myopic guinea pigs, which exaggerated fibrotic lesions and reduced retinal thickness, ultimately leading to retinal physiological dysfunctions in myopic guinea pigs.

Protective Effects of Phytic Acid on CCl4-Induced Liver Fibrosis in Mice

Liver fibrosis is the main pathological feature of various chronic liver diseases that can progress to cirrhosis. However, no effective treatment strategy for liver fibrosis is available. Phytic acid (PA) is a natural plant compound found in cereals, legumes, and rapeseed. Currently, there are few reports on its relationship with liver fibrosis. Herein, we explored the effects of PA on liver fibrosis. In this study, the liver fibrosis model was constructed by intraperitoneal injection of CCl4 and intragastric administration of sodium phytate (100 mg/kg) or silymarin (100 mg/kg) five times a week. The CCl4 injection could significantly increase the content of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and induce the damage of liver cells, infiltration of inflammatory factors, and deposition of collagen fibers in the liver tissue. Compared to the CCl4 group, PA significantly reduced AST and ALT. Also, PA alleviated liver damage and reduced inflammatory cell infiltration, collagen deposition area, and hydroxyproline (Hyp) content in the liver tissue, as well as downregulated α-smooth muscle actin (α-SMA) and collagen I (Col-1). Mechanistically, PA downregulated PI3K, p-AKT, p-p65, and p-IκBα in the liver tissue and reduced the release of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). In conclusion, PA might ameliorate liver fibrosis by inhibiting the NF-κB and PI3K/AKT signaling pathways.

In vivo Performance of a Hydrogel of Tribulus terrestris in Alloxan induced Diabetic Rats

Abstract: Background: Diabetes is one of the most important factors in chronic injuries. The gold standard for treating diabetic foot ulcers includes debridement of wounds, management of all infections, revascularization when indicated, and non-loading of ulcers. Traditionally, the aerial parts of Tribulus terrestris L. (Zygophyllaceae) are used in the folklore for the treatment of various kinds of wounds. Materials and Methods: Based on the evaluation of phytochemicals, a hydrogel of the hydroalcoholic and the aqueous of T. terrestris were prepared in this study. It was subjected to evaluation of the different parameters. The hydrogel of hydroalcoholic extract and aqueous extract was brown in color, uniform and non-irritant. It was further evaluated for pH, spreadability and viscosity. Furthermore, the in vivo performance of the hydrogel was evaluated in alloxan monohydrate induced diabetic rats. Results: In excision wound model and incision wound model topical application of 5% hydroalcoholic extract hydrogel in diabetic Wistar albino rat showed remarkable repair effects. It significantly (***p<0.001) enhanced the rate of % wound contraction. It also exhibited significant (***p<0.001) content of hydroxyproline and extremely high tensile strength (***p<0.001) as compared to the diabetic control group. It may be due to enhanced collagen synthesis. The histopathological examination of 5% hydroalcoholic extract hydrogel of T. terrestris L. treated rats showed skin architecture which was comparable to the normal group. The diabetes-induced group rats’ wounds failed to heal even 25 days post the infliction of the wounds. Our model precisely reproduced the pathophysiology of diabetic ulcers and can serve as a valid alternative model for diabetic foot ulcer and to explore new therapies. Conclusion: The results strongly support that the hydrogel of the hydroalcoholic extract of T. terrestris seems to be a novel and promising biomaterial used for wound healing applications. Keywords: Diabetes, Tribulus terrestris, Wound healing, Alloxan, Hydroxyproline, Hydrogel.

Targeted imaging of very late antigen-4 for noninvasive assessment of lung inflammation-fibrosis axis

BackgroundThe lack of noninvasive methods for assessment of dysregulated inflammation as a major driver of fibrosis (i.e., inflammation-fibrosis axis) has been a major challenge to precision management of fibrotic lung diseases. Here, we determined the potential of very late antigen-4 (VLA-4)-targeted positron emission tomography (PET) to detect inflammation in a mouse model of bleomycin-induced fibrotic lung injury.MethodSingle time-point and longitudinal VLA-4-targeted PET was performed using a high-affinity peptidomimetic radiotracer, 64Cu-LLP2A, at weeks 1, 2, and 4 after bleomycin-induced (2.5 units/kg) lung injury in C57BL/6J mice. The severity of fibrosis was determined by measuring the hydroxyproline content of the lungs and expression of markers of extracellular matrix remodeling. Flow cytometry and histology was performed to determine VLA-4 expression across different leukocyte subsets and their spatial distribution.ResultsLung uptake of 64Cu-LLP2A was significantly elevated throughout different stages of the progression of bleomycin-induced injury. High lung uptake of 64Cu-LLP2A at week-1 post-bleomycin was a predictor of poor survival over the 4-week follow up, supporting the prognostic potential of 64Cu-LLP2A PET during the early stage of the disease. Additionally, the progressive increase in 64Cu-LLP2A uptake from week-1 to week-4 post-bleomycin correlated with the ultimate extent of lung fibrosis and ECM remodeling. Flow cytometry revealed that LLP2A binding was restricted to leukocytes. A combination of increased expression of VLA-4 by alveolar macrophages and accumulation of VLA-4-expressing interstitial and monocyte-derived macrophages as well as dendritic cells was noted in bleomycin-injured, compared to control, lungs. Histology confirmed the increased expression of VLA-4 in bleomycin-injured lungs, particularly in inflamed and fibrotic regions.ConclusionsVLA-4-targeted PET allows for assessment of the inflammation-fibrosis axis and prediction of disease progression in a murine model. The potential of 64Cu-LLP2A PET for assessment of the inflammation-fibrosis axis in human fibrotic lung diseases needs to be further investigated.

Assessment of Hydroxyproline Content in Rabbit Achilles Tendon Treated with Platelet Rich Fibrin (PRF).

Tendon is similar to rope and consists of strong, flexible, dense connective tissue. Tendon disorder healing is challenging as it is an avascular tissue. The repaired tissue appears scar-like, and its biomechanical properties never ultimately return to their pre-injury state. This study aimed to evaluate the effects of platelet-rich fibrin (PRF) on the hydroxyproline content of the Achilles tendon after injury. For this purpose, 24 adult rabbits weighing 1.5-2 kg were used in this study. The animals were divided into three groups of eight, including the advanced-PRF (A-PRF) group in which the tendon defect was treated with xenogeneic A-PRF, the leukocyte-PRF (L-PRF) group in which xenogeneic L-PRF was used for tendon defect treatment, and a control group which was treated with normal saline. Hydroxyproline concentration was measured 1 and 2 months after the operation. Clinically, lameness was improved in the A-PRF group, compared to the L-PRF and control groups at the end of the third week after the surgery. Hydroxyproline level was significantly increased in the A-PRF group (50.33±1.44), compared to the L-PRF (44.70±1.12) and control (35.97±1.05) groups 2 months after the surgery (P<0.05). Moreover, the L-PRF group showed an increase in hydroxyproline content, compared to the control at the same period. The results of the current study demonstrated that A-PRF could enhance the hydroxyproline content of rabbit Achilles tendon after injury. Xenogenic PRF can be used as an alternative biomaterial to accelerate and regenerate tendon tissue.

Sparganii Rhizoma alleviates pulmonary fibrosis by inhibiting fibroblasts differentiation and epithelial-mesenchymal transition mediated by TGF-β1/ Smad2/3 pathway.

Pulmonary fibrosis (PF), a lethal lung disease, can lead to structural destruction of the alveoli until death. Sparganii Rhizoma (SR), primarily distributed in East Asia, has been used clinically for hundreds of years against organ fibrosis and inflammation. We intended to verify the effect of SR alleviate PF and further explore mechanisms. Murine model of PF was established by endotracheal infusion of bleomycin. We detected the anti-PF effect of SR through lung coefficient, hydroxyproline content, lung function and pathological staining. Then, we used Western Blot and RT-PCR to verify the mechanism. In vitro experiments, MRC-5 and BEAS-2B were induced to phenotypic transformation by TGF-β1 and then RT-PCR, WB and IF were conducted to verify the effect of SR. SR significantly reduced BLM-induced PF in mice, improved lung function, slowed the degree of lung tissue lesions, and reduced collagen deposition. SR alleviated PF by inhibiting fibroblasts differentiation and epithelial-mesenchymal transition. In vivo studies explored the mechanism and found that it was related to TGF-β1/Smad2/3 pathway. Our research proved SR could effectively treat PF, providing a fresh idea and approach for the treatment of PF with traditional Chinese medicine.

Empagliflozin Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats by Modulating Sesn2/AMPK/Nrf2 Signaling and Targeting Ferroptosis and Autophagy.

Pulmonary fibrosis (PF) is a life-threatening disorder that severely disrupts normal lung architecture and function, resulting in severe respiratory failure and death. It has no definite treatment. Empagliflozin (EMPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, has protective potential in PF. However, the mechanisms underlying these effects require further elucidation. Therefore, this study aimed to evaluate the ameliorative effect of EMPA against bleomycin (BLM)-induced PF and the potential mechanisms. Twenty-four male Wister rats were randomly divided into four groups: control, BLM treated, EMPA treated, and EMPA+BLM treated. EMPA significantly improved the histopathological injuries illustrated by both hematoxylin and eosin and Masson's trichrome-stained lung tissue sections, as confirmed by electron microscopic examination. It significantly reduced the lung index, hydroxyproline content, and transforming growth factor β1 levels in the BLM rat model. It had an anti-inflammatory effect, as evidenced by a decrease in the inflammatory cytokines' tumor necrosis factor alpha and high mobility group box 1, inflammatory cell infiltration into the bronchoalveolar lavage fluid, and the CD68 immunoreaction. Furthermore, EMPA mitigated oxidative stress, DNA fragmentation, ferroptosis, and endoplasmic reticulum stress, as evidenced by the up-regulation of nuclear factor erythroid 2-related factor expression, heme oxygenase-1 activity, glutathione peroxidase 4 levels, and a decrease in C/EBP homologous protein levels. This protective potential could be explained on the basis of autophagy induction via up-regulating lung sestrin2 expression and the LC3 II immunoreaction observed in this study. Our findings indicated that EMPA protected against BLM-induced PF-associated cellular stress by enhancing autophagy and modulating sestrin2/adenosine monophosphate-activated protein kinase/nuclear factor erythroid 2-related factor 2/heme oxygenase 1 signaling.

Protein corona formed on the TiO2 nanoparticles promotes the hydrolysis of collagen in simulated gastrointestinal fluids

Titanium dioxide nanoparticles (TiO2 NPs) could interact with proteins in foods and gastrointestinal fluids, thus changing the properties and functions of proteins. Many research articles have only studied the interactions between TiO2 NPs and digestive enzymes or the gastrointestinal fate of TiO2 NPs without considering the effect of food matrix. Therefore, the interactions between TiO2 NPs and trypsin and its effects on the digestion of collagen in vitro were investigated. Results showed that the functionalization of amino acids (Asp, and Ser) significantly harmed the adsorption of trypsin by TiO2 NPs. The fluorescence quenching and isothermal titration calorimetry confirmed that the hydrogen bond and van der Waals force played an essential role in the interactions between TiO2 NPs and trypsin. The β-sheet content of trypsin bound to TiO2 NPs decreased from 52.40% to 50.73% while the random coil content increased from 20.20% to 22.63%. Binding sites were identified by molecular docking. Additionally, the digestibility of collagen was improved in vitro in the presence of TiO2 NPs analyzed by SDS-PAGE, hydroxyproline quantification, and the antioxidation of digested products. Hydroxyproline concentration increased by nearly 25% and ABTS radical scavenging rate increased by 50%. This study provides a new insight into the impact of nanoparticles on gastrointestinal digestion.

Changes in the indicators of connective tissue metabolism in the blood serum of experimental rats under the conditions of modeling the development of degenerative processes in paravertebral muscles

Low back pain is a common health problem. To deepen the understanding of the pathogenesis of the disease, experimental studies on animals with modeling of the pathological process are necessary. Objective. Based on the analysis of biochemical markers of connective tissue metabolism in the blood serum of laboratory rats, the applicability of the studied models of degenerative muscle tissue damage to study the relationship between this condition and the development of disorders in spinal motor segments was assessed. Methods. Two models of reproduction of degenerative processes in the paravertebral muscles of white rats were tested: I (n = 5) — alimentary (diet-induced) obesity, by keeping it for 3 months on a high-calorie diet; II (n = 5) — ischemia, by tying the large rectus muscles of the back with suture material (45 days). Control group (n = 5) — intact animals of similar age and sex. The content of glycoproteins, total chondroitin sulfates (CS), hexosamines, protein-bound hexoses, seroglycoides, fractional distribution and total content of hydroxyproline and glycosaminoglycan sulfates (GAGs) were investigated in the blood serum of rats. The results. In the blood serum of rats of groups I and II, a significant increase compared to the control level of glycoproteins was determined, with a greater effect in the ischemia model, but no significant changes of protein-bound hexoses, hexosamines and CS were recorded. The level of inflammatory markers (sialic acids and seroglycoides) in the blood serum of animals of both groups did not differ significantly from the control, and changes in the parameters of hydroxyproline (except for the slightly changed protein-bound fraction) and GAGs were significant only for the ischemia model. Conclusions. Based on the analysis of biochemical markers of connective tissue metabolism in rats of groups I and II, changes characteristic of degenerative processes were determined, with a greater manifestation in the ischemia model. No significant increase in biochemical markers of inflammation was recorded. Both models can be used to reproduce dystrophic processes in osteochondrosis.

Effect of Ultrasonic Amplitude on the Yield and Properties of Barramundi (Lates calcarifer) Skin Collagen

Barramundi skin, a by-product of the fish processing industry, has shown potential as an alternative collagen source. However, the commonly used acid extraction method to produce collagen rendered a low yield requires a lengthy time and is not environmentally friendly. As a result, the adoption of greener technology, such as ultrasound, to improve the conventional extraction process is emerging. This study aimed to investigate the effect of different ultrasonication amplitudes on collagen recovery from barramundi skin. The resulting collagens were evaluated for their protein, hydroxyproline and moisture content, colour, molecular weight distribution, and FTIR spectra. Ultrasound-assisted extraction (UAE) was performed at 40 (UAE40), 60 (UAE60) and 80 (UAE80) % amplitude for 20 min. For comparison, acetic acid extraction was also carried out to produce acid-soluble collagen (ASC). UAE increased the yield (p&lt;0.05) of collagen from barramundi skin, with UAE80 exhibiting a 7-fold increment compared to ASC. Increasing the ultrasonic amplitude increased the yield considerably but decreased the hydroxyproline content, indicating a reduction in collagen quality. Furthermore, the protein content and SDS-PAGE profile of the extracted collagens revealed that UAE promoted protein degradation. FTIR spectra indicated that despite slightly varying wavenumbers, no detrimental effect on the triple helical structure was seen following UAE with the presence of amides A, B, I, II, and III. Also, the α1, α2 and ß-chains were found in all samples, although the band intensity reduced as the amplitude increased. In conclusion, given the right conditions, UAE could improve the extraction yield without influencing the collagen structure.

Schizandrin C regulates lipid metabolism and inflammation in liver fibrosis by NF-κB and p38/ERK MAPK signaling pathways.

Liver fibrosis is considered a sustained wound healing response and metabolic syndrome, and its therapy is of great significance for chronic liver disease. Schizandrin C, as one lignan from hepatic protectant Schisandra chinensis, can depress the oxidative effect and lipid peroxidation, and protect against liver injury. In this study, C57BL/6J mice were used to estimate a liver fibrosis model by CCl4, and Schizandrin C exerted an anti-hepatic fibrosis effect, as evidenced by decreased alanine aminotransferase, aspartate aminotransferase and total bilirubin activities in serum, lower hydroxyproline content, recuperative structure and less collagen accumulation in the liver. In addition, Schizandrin C reduced the expressions of alpha-smooth muscle actin and type Ι collagen in the liver. In vitro experiments also revealed that Schizandrin C attenuated hepatic stellate cell activation in both LX-2 and HSC-T6 cells. Furthermore, lipidomics and quantitative real-time PCR analysis revealed that Schizandrin C regulated the lipid profile and related metabolic enzymes in the liver. In addition, the mRNA levels of inflammation factors were downregulated by Schizandrin C treatment, accompanied by lower protein levels of IκB-Kinase-β, nuclear factor kappa-B p65, and phospho-nuclear factor kappa-B p65. Finally, Schizandrin C inhibited the phosphorylation of p38 MAP kinase and extracellular signal-regulated protein kinase, which were activated in the CCl4 fibrotic liver. Taken together, Schizandrin C can regulate lipid metabolism and inflammation to ameliorate liver fibrosis by nuclear factor kappa-B and p38/ERK MAPK signaling pathways. These findings supported Schizandrin C as a potential drug for liver fibrosis.