mRNA Expression Of Collagen Type Research Articles

SPARC: A MODULATOR OF TENDON HOMEOSTASIS AND HEALING

Tendons are characterised by an inferior healing capacity when compared to other tissues, ultimately resulting in the formation of a pathologically altered extracellular matrix structure. Although our understanding of the underlying causes for the development and progression of tendinopathies remains incomplete, mounting evidence indicates a coordinated interplay between tendon-resident cells and the ECM is critical. Our recent results demonstrate that the matricellular protein SPARC (Secreted protein acidic and rich in cysteine) is essential for regulating tendon tissue homeostasis and maturation by modulating the tissue mechanical properties and aiding in collagen fibrillogenesis [1,2]. Consequently, we speculate that SPARC may also be relevant for tendon healing.In a rat patellar tendon window defect model, we investigated whether the administration of recombinant SPARC protein can modulate tendon healing. Besides the increased mRNA expression of collagen type 1 and the downregulation of collagen type 3, a robust increase in the expression of pro-regenerative fibroblast markers in the repair tissue after a single treatment with rSPARC protein was observed. Additionally, pro-fibrotic markers were significantly decreased by the administration of rSPARC. Determination of structural characteristics was also assessed, indicating that the ECM structure can be improved by the application of rSPARC protein. Therefore, we believe that SPARC plays an important role for tendon healing and the application of recombinant SPARC to tendon defects has great potential to improve functional tendon repair.

Niclosamide Attenuates Inflammation-Associated Profibrotic Responses in Human Subepithelial Lung Myofibroblasts.

Niclosamide is a commonly used helminthicidic drug for the treatment of human parasitosis by helminths. Recently, efforts have been focusing on repurposing this drug for the treatment of other diseases, such as idiopathic pulmonary fibrosis. Subepithelial lung myofibroblasts (SELMs) isolated from tissue biopsies of patients undergoing surgery for lung cancer were stimulated with TNF-α (50 ng/mL), IL-1α (5 ng/mL), added alone or in combination, and TGF-β1 (5 ng/mL). After treatment with niclosamide at 30 nM and 100 nM concentrations, expression of collagen type I, collagen type III, and fibronectin was studied by total RNA isolation and qRT-PCR and protein collagen secretion with the use of Sircol collagen assay. The migration of SELMs was assessed by a wound-healing assay. Niclosamide had no effect on baseline SELM fibrotic factor expression. When stimulated with TGF-β1, IL-1α, and/or TNF-α, SELM expression of collagen type I, type III, and fibronectin were upregulated, as was the secretion of total collagen in the culture medium. Treatment with niclosamide attenuated the effects of cytokine stimulation leading to a notable decrease in the mRNA expression of collagen type I, type III, and fibronectin in a concentration-dependent manner. SELM collagen secretion was also reduced by niclosamide at 100 nM concentration when examined at the protein level. Migration of both TGF-β1 stimulated and unstimulated SELMs was also inhibited by niclosamide. In this study, we highlight the anti-fibrotic properties of niclosamide on SELMs under stimulation with pro-fibrotic and pro-inflammatory cytokines, thus proposing this compound as a possible new therapeutic agent against lung fibrosis.

Bioactive Scaffold With Spatially Embedded Growth Factors Promotes Bone-to-Tendon Interface Healing of Chronic Rotator Cuff Tear in Rabbit Model

Background: Functional restoration of the bone-to-tendon interface (BTI) after rotator cuff repair is a challenge. Therefore, numerous biocompatible biomaterials for promoting BTI healing have been investigated. Purpose: To determine the efficacy of scaffolds with spatiotemporal delivery of growth factors (GFs) to accelerate BTI healing after rotator cuff repair. Study Design: Controlled laboratory study. Methods: An advanced 3-dimensional printing technique was used to fabricate bioactive scaffolds with spatiotemporal delivery of multiple GFs targeting the tendon, fibrocartilage, and bone regions. In total, 50 rabbits were used: 2 nonoperated controls and 48 rabbits with induced chronic rotator cuff tears (RCTs). The animals with RCTs were divided into 3 groups: (A) saline injection, (B) scaffold without GF, and (C) scaffold with GF. To induce chronic models, RCTs were left unrepaired for 6 weeks; then, surgical repairs with or without bioactive scaffolds were performed. For groups B and C, each scaffold was implanted between the bony footprint and the supraspinatus tendon. Four weeks after repair, quantitative real-time polymerase chain reaction and immunofluorescence analyses were performed to evaluate early signs of regenerative healing. Histological, biomechanical, and micro–computed tomography analyses were performed 12 weeks after repair. Results: Group C had the highest mRNA expression of collagen type I alpha 1, collagen type III alpha 1, and aggrecan. Immunofluorescence analysis showed the formation of an aggrecan+/collagen II+ fibrocartilaginous matrix at the BTI when repaired with scaffold with GFs. Histologic analysis revealed greater collagen fiber continuity, denser collagen fibers, and a more mature tendon-to-bone junction in GF-embedded scaffolds than those in the other groups. Group C demonstrated the highest load-to-failure ratio, and modulus mapping showed that the distribution of the micromechanical properties of the BTI repaired with GF-embedded scaffolds was comparable with that of the native BTI. Micro–computed tomography analysis identified the highest bone mineral density and bone volume/total volume ratio in group C. Conclusion: Bioactive scaffolds with spatially embedded GFs have significant potential to promote the BTI healing of chronic RCTs in a rabbit model. Clinical Relevance: The scaffolds with spatiotemporal delivery of GF may serve as an off-the-shelf biomaterial graft to promote the healing of RCTs.

Human amnion epithelial cells reduces hypertension induced vascular stiffening and cognitive impairment

Introduction: Vascular stiffening and inflammation are hallmarks of hypertension and contribute to the development of cardiovascular disease and cognitive impairment. Current treatments for hypertension may reduce blood pressure but do not target the associated pathological changes and end-organ damage. It is also inconclusive from clinical trials whether anti-hypertensive medication can improve cognitive impairment. Cell therapy has great therapeutic potential as unlike single pharmacological agents, cells can deliver multiple mediators which could more effectively target complex disease mechanisms. Human amnion epithelial cells (AECs) have many properties (eg. anti-inflammatory, anti-fibrotic, and immunologically inert) that make them attractive candidates for a cell-based therapy for vascular and brain pathology. Aim: To test the potential of AECs to treat vascular and brain pathology in angiotensin II-induced hypertension. Methods. Male C57Bl6 mice (8-12 weeks) were administered vehicle (saline; n=35) or angiotensin II (0.7 mg/kg/d, n=37) for 14 d via an osmotic minipump. After minipump implantation, a subset of mice were injected with 10 6 of AECs intravenously. Systolic blood pressure was measured using tail-cuff; pulse wave velocity was measured using ultrasound, inflammation was assessed using flow cytometry and RNA sequencing, markers of fibrosis using quantitative PCR and cognitive function using novel object recognition test. Results: Angiotensin II infusion increased blood pressure and aortic pulse wave velocity (n=6-7, P<0.05). In aorta, angiotensin II promoted accumulation of leukocytes, specifically macrophages and monocytes, which were elevated by ~3-fold compared to vehicle-infused mice (n=9-11, P<0.05). Angiotensin II also increased aortic mRNA expression of collagen type 1 alpha 1 ( Col1a1) by ~4-fold and collagen type 5 alpha 1 ( Col5a1) by ~7-fold compared to vehicle (n=6-8, P<0.05). Co-administration of AECs limited the development of hypertension and aortic stiffening by angiotensin II (185±5 mmHg vs 165±4 mmHg; n=9-11, P<0.05), as well as the aortic infiltration of macrophages and monocytes (n=9-11, P<0.05) and expression of Col1a1 and Col5a1 (n=6-8, P<0.05). Infusion with angiotensin II also impaired memory which was improved by AECs (n=7-9, P<0.05). RNA sequencing revealed that in the brain, angiotensin II increased expression of genes involved in inflammation ( Ptcra, Blk, Bpifb1), fibrosis ( Col6a5) and apoptosis ( Fas) and co-administration of AECs caused downregulation of these genes. Conclusions: Intravenous administration of AECs blunted angiotensin II-induced hypertension, aortic stiffening, inflammation and cognitive impairment in male mice. The beneficial effects of AECs on blood pressure and aortic stiffening could have contributed to the improvement in cognition. This study suggests that AECs or their cellular products could be explored as treatments for vascular and brain pathology during hypertension. La Trobe University Start Up grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anti-Oxidative and Anti-Aging Effects of Ethanol Extract of the Officinal Breynia (Breynia vitis-idaea) In Vitro.

The skin is the largest organ of the human body, and it is also the one most exposed to external environmental contaminants. The skin is the body's first defense against harmful environmental stimuli, including ultraviolet B (UVB) rays and hazardous chemicals. Therefore, proper care of the skin is required to prevent skin-related diseases and age-related symptoms. In this study, we analyzed anti-aging and anti-oxidative effects of Breynia vitis-idaea ethanol extract (Bv-EE) in human keratinocytes and dermal fibroblasts. The Bv-EE had free radical scavenging activity and decreased the mRNA expression of MMPs and COX-2 in H2O2- or UVB-treated HaCaT cells. The Bv-EE also inhibited AP-1 transcriptional activity and phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and mitogen-activated protein kinase 14 (p38), which are major AP-1 activators upon H2O2 or UVB exposure. Furthermore, the promoter activity and mRNA expression of collagen type I (Col1A1) increased in HDF cells treated with Bv-EE, and Bv-EE recovered the collagen mRNA expression decreased by H2O2 or UVB exposure. These results suggest that Bv-EE has anti-oxidative effects by inhibiting the AP-1 signaling pathway, and shows anti-aging effects by upregulating collagen synthesis.

Hepatoprotective Efficacy of Cycloastragenol Alleviated the Progression of Liver Fibrosis in Carbon-Tetrachloride-Treated Mice.

The continuous death of hepatocytes induced by various etiologies leads to an aberrant tissue healing process and promotes the progression of liver fibrosis and ultimately chronic liver diseases. To date, effective treatments to delay this harmful process remain an unmet clinical need. Cycloastragenol is an active phytochemical substance isolated from Astragalus membranaceus, a plant used in traditional Chinese medicine to protect the liver. Therefore, our study aimed to elucidate the efficacy of cycloastragenol on carbon-tetrachloride (CCl4)-induced liver fibrosis in mice. We found that cycloastragenol at 200 mg/kg dosage exhibited anti-fibrotic efficacy as demonstrated by a decrease in collagen deposition, downregulation of mRNA expression of collagen type 1, and a reduction in the content of total collagens. In addition, cycloastragenol further augmented the levels of anti-fibrotic matrix metalloproteinases (Mmps), that is, Mmp8, proMmp9, and Mmp12, which play a pivotal role in fibrosis resolution. According to histological analysis and serum markers of hepatotoxicity, cycloastragenol protected the livers from damage and mitigated the increment of serum alanine aminotransferase and bilirubin implicating hepatoprotective efficacy against CCl4. Moreover, cycloastragenol upregulated the mRNA expression of interleukin 6, a pleiotropic cytokine plays a vital role in the promotion of hepatocyte regeneration. In conclusion, cycloastragenol alleviated the progression of liver fibrosis in CCl4-treated mice and its anti-fibrotic efficacy was mainly due to the hepatoprotective efficacy.

Amentoflavone-Enriched Selaginella rossii Protects against Ultraviolet- and Oxidative Stress-Induced Aging in Skin Cells.

Selaginellaceae plants are used in cosmetics to limit skin aging. This study is the first to investigate the anti-aging effects of Selaginella rossii (SR) on ultraviolet B (UVB)- and oxidative stress-induced skin cells. The 95% ethanol extract of Selaginella rossii (SR95E) contained much higher amounts of amentoflavone (AMF), an active compound, than other Selaginellaceae plants and was more effective in inhibiting matrix metalloproteinase (MMP)-1 expression in CCD-986sk fibroblasts. SR95E significantly decreased UVB-induced MMP-1, MMP-2, MMP-3 and MMP-9 expression and enhanced procollagen type I C-peptide content and mRNA expression of collagen type I alpha (COL1A)1 and COL1A2 in CCD-986sk fibroblasts. In HaCaT keratinocytes, SR95E treatment also dose-dependently decreased UVB-induced MMP-1 concentration and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA expression. Moreover, SR95E treatment markedly inhibited UVB-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling and nuclear factor kappa-B signaling in HaCaT cells. Furthermore, SR95E and AMF markedly regulated the 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced expression of cellular senescence-related markers, including p16, p21 and LMNB1, in HaCaT cells. Overall, this study indicates that SR may have potential as a functional material on preventing UVB- and AAPH-induced skin aging and wrinkles.

Mechanism of Akt regulation of the expression of collagens and MMPs in conjunctivochalasis

Akt is a central node of many signaling pathways, which plays important roles in cell survival, proliferation, migration, metabolism and collagen synthesis. Conjunctivochalasis (CCH) is one of the most common age-related ocular superficial diseases related to abnormalities in conjunctival extracellular matrix. Here, we studied the role of Akt regulating collagens and MMPs in the pathogenesis of CCH. Primary conjunctival fibroblasts were obtained from CCH patients (n = 13) and age-matched normal controls (n = 10). The levels of Akt, collagen type I, collagen type III, MMP1, and MMP3 were determined by Western blot, qRT-PCR, immunohistochemistry, and immunofluorescence staining. Normal control conjunctival fibroblasts were treated with Akt inhibitor A6730, and CCH fibroblasts were transfected with Akt overexpression vector. The expression of Akt in CCH was significantly lower than that in normal control of conjunctival tissues and cultured fibroblasts. Blocking Akt signaling with Akt inhibitor could inhibit the expression of collagen type I and collagen type III and upregulate the expression of MMP1 and MMP3. Meanwhile, compared with CCH fibroblasts transfected with control mimics, the protein and mRNA expression of collagen type I and collagen type III were increased significantly in Akt overexpression group, while the results of MMP1 and MMP3 in transfected fibroblasts were opposite. Taken together, Akt upregulated the expression of collagen type I and collagen type III and downregulated the expression of MMP1 and MMP3. Akt signaling pathway could provide a direct negative contribution to CCH and might be an attractive target for CCH therapy.

Inhibition of FABP4 attenuates cardiac fibrosis through inhibition of NLRP3 inflammasome activation.

Objective(s):Cardiac fibrosis is a key biological process of cardiac remodeling and heart failure. Fatty acid-binding protein 4 (FABP4) is a lipid-binding protein that can regulate glucose and lipid homeostasis, and its expression was elevated in heart failure. However, whether FABP4 is involved in cardiac fibrosis remains unknown. Materials and Methods:The cardiac fibrosis model was established in male C57BL/6 mice with subcutaneously infused angiotensin II (Ang-II) (2.8 mg/kg/day) for 4 weeks. DMSO or FABP4 inhibitor BMS309403 (50 mg/kg/day) was intraperitoneally injected for 4 weeks. Ang II-infused mice, FABP4 inhibitor (BMS309403) injected mice, and ventricular tissue were used for morphological studies, and histological and biochemical analyses (FABP4 protein composition and expression).Results:Ang II infusion increased FABP4 mRNA and protein expression in the mouse ventricular tissue. After treatment with FABP4 inhibitor BMS309403 for 4 weeks, mice showed improved cardiac structure and function as detected by echocardiography. BMS309403 suppressed cardiac and systemic inflammatory response, reduced collagen deposition, and mRNA expression of collagen type I (COL1A1) and collagen type III (COL3A1) in Ang II-infused mice. BMS309403 also reduced the number of α-smooth muscle actin (α-SMA)+cells and decreased the mRNA expression of α-SMA, matrix metalloproteinases-2 (MMP-2), MMP-9, and transforming growth factor-β (TGFβ) in ventricular tissue.Conclusion:The inhibitory effect of BMS309403 on cardiac fibrosis might be associated with inhibition of NLRP3 inflammasome activation, which Ang II activated. Thus, our data speculated that inhibition of FABP4 could significantly induce cardiac fibrosis.

Enhanced healing efficacy of an optimized gabapentin-melittin nanoconjugate gel-loaded formulation in excised wounds of diabetic rats

The present study aimed to design and optimize, a nanoconjugate of gabapentin (GPN)-melittin (MLT) and to evaluate its healing activity in rat diabetic wounds. To explore the wound healing potency of GPN-MLT nanoconjugate, an in vivo study was carried out. Diabetic rats were subjected to excision wounds and received daily topical treatment with conventional formulations of GPN, MLT, GPN-MLT nanoconjugate and a marketed formula. The outcome of the in vivo study showed an expedited wound contraction in GPN-MLT-treated animals. This was confirmed histologically. The nanoconjugate formula exhibited antioxidant activities as evidenced by preventing malondialdehyde (MDA) accumulation and superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymatic exhaustion. Further, the nanoconjugate showed superior anti-inflammatory activity as it inhibited the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). This is in addition to enhancement of proliferation as indicated by increased expression of transforming growth factor-β (TGF- β), vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor receptor-β (PDGFRB). Also, nanoconjugate enhanced hydroxyproline concentration and mRNA expression of collagen type 1 alpha 1 (Col 1A1). In conclusion, a GPN-MLT nanoconjugate was optimized with respect to particle size. Analysis of pharmacokinetic attributes showed the mean particle size of optimized nanoconjugate as 156.9 nm. The nanoconjugate exhibited potent wound healing activities in diabetic rats. This, at least partly, involve enhanced antioxidant, anti-inflammatory, proliferative and pro-collagen activities. This may help to develop novel formulae that could accelerate wound healing in diabetes.

Anti-Inflammatory and Anti-Fibrotic Effect of Immortalized Mesenchymal-Stem-Cell-Derived Conditioned Medium on Human Lung Myofibroblasts and Epithelial Cells.

Idiopathic pulmonary fibrosis (IPF) is caused by progressive lung tissue impairment due to extended chronic fibrosis, and it has no known effective treatment. The use of conditioned media (CM) from an immortalized human adipose mesenchymal stem cell line could be a promising therapeutic strategy, as it can reduce both fibrotic and inflammatory responses. We aimed to investigate the anti-inflammatory and anti-fibrotic effect of CM on human pulmonary subepithelial myofibroblasts (hPSM) and on A549 pulmonary epithelial cells, treated with pro-inflammatory or pro-fibrotic mediators. CM inhibited the proinflammatory cytokine-induced mRNA and protein production of various chemokines in both hPSMs and A549 cells. It also downregulated the mRNA expression of IL-1α, but upregulated IL-1β and IL-6 mRNA production in both cell types. CM downregulated the pro-fibrotic-induced mRNA expression of collagen Type III and the migration rate of hPSMs, but upregulated fibronectin mRNA production and the total protein collagen secretion. CM’s direct effect on the chemotaxis and cell recruitment of immune-associated cells, and its indirect effect on fibrosis through the significant decrease in the migration capacity of hPSMs, makes it a plausible candidate for further development towards a therapeutic treatment for IPF.

Human outgrowth knee fibroblasts from patients undergoing total knee arthroplasty exhibit a unique gene expression profile and undergo myofibroblastogenesis upon TGFβ1 stimulation.

Arthrofibrosis is characterized by excessive extracellular matrix (ECM) deposition that results in restricted joint motion after total knee arthroplasties (TKAs). Currently, treatment options are limited. Therefore, an in vitro model of knee-related myofibroblastogenesis is valuable to facilitate investigation of the arthrofibrotic process, diagnostic and therapeutic options. In this study, we obtained intraoperative posterior capsule (PC), quadriceps tendon (QT), and suprapatellar pouch (SP) tissues from the knees of four patients undergoing primary TKAs for osteoarthritis. From these tissues, we isolated primary cells by the outgrowth method and subsequently characterized these cells in the absence and presence of the pro-myofibroblastic cytokine, transforming growth factor beta 1 (TGFβ1). Light microscopy of knee outgrowth cells revealed spindle-shaped cells, and immunofluorescence (IF) analysis demonstrated staining for the fibroblast-specific markers TE-7 and vimentin (VIM). These knee outgrowth fibroblasts differentiated readily into myofibroblasts as reflected by enhanced α-smooth muscle actin (ACTA2) mRNA and protein expression and increased mRNA expression of collagen type 1 (COL1A1) and type 3 (COL3A1) with collagenous matrix deposition in the presence of TGFβ1. Outgrowth knee fibroblasts were more sensitive to TGFβ1-mediated myofibroblastogenesis than adipose-derived mesenchymal stromal/stem cells (MSCs). While outgrowth knee fibroblasts isolated from three anatomical regions in four patients exhibited similar gene expression, these cells are distinct from other fibroblastic cell types (i.e., Dupuytren's fibroblasts) as revealed by RNA-sequencing. In conclusion, our study provides an in vitro myofibroblastic model of outgrowth knee fibroblasts derived from patients undergoing primary TKA that can be utilized to study myofibroblastogenesis and assess therapeutic strategies for arthrofibrosis.

P093 Probiotic strains Lactobacillus plantarum and Saccharomyces boulardii influence the fibrotic responses of human intestinal subepithelial myofibroblasts

Abstract Background The strains Lactobacillus plantarum and Saccharomyces boulardii, widely used among probiotic supplements, seem to ameliorate Inflammatory Bowel Disease (IBD) progression and to promote mucosal healing. Intestinal Subepithelial Myofibroblasts (SEMFs) are key mediators in mucosal healing and fibrosis, mainly by migrating to the trauma region and secreting collagen. The aim of this study was to investigate the different effects of the aforementioned probiotic strains in the migration rate and the fibrotic responses of SEMFs. Methods L. plantarum and S. boulardii were reconstituted in SEMFs culture medium, identified by Gram staining and their viability was assessed by Trypan Blue staining. Primary SEMFs were isolated from colonic biopsies from healthy individuals and stimulated with 102 and 104 cfu/ml of each strain for: a) 6h, when mRNA expression of collagen type I, III, fibronectin, Tissue Factor and α-SMA was assessed by quantitative PCR, and b) 48h, when SEMFs’ migration rate was examined via Wound Healing Assay and protein collagen was measured via Sircol Assay. Results Trypan Blue staining confirmed the viability of the studied probiotics, and both the bacteria and the yeasts were stained Gram positive. Stimulation of SEMFs with L. plantarum resulted in upregulated mRNAs of Collagen type I (102cfu/ml: 1.6-fold, 104cfu/ml: 1.5-fold; p<0.001), type III (102cfu/ml: 1.7-fold, 104cfu/ml: 1.8-fold; p<0.0001), Fibronectin (102 cfu/ml, 1.5-fold, 104cfu/ml 1.5-fold; p<0.01), Tissue Factor (102cfu/ml: 1.4-fold, p<0.0001; 104cfu/ml: 1.3-fold, p<0.001) as well as SEMFs’ migration rate (102cfu/ml: 128.3%, 104cfu/ml: 124.1%; p<0.05). On the other hand, S. boulardii stimulation resulted in downregulated Collagen type I (102cfu/ml: 0.9-fold, p<0.01) and type III mRNAs (102cfu/ml: 0.8-fold, p<0.01), upregulated Tissue Factor mRNA (102cfu/ml: 1.2-fold, p<0.0001; 104cfu/ml: 1.1-fold, p<0.01), increased protein collagen production (104cfu/ml: by 211.6%, p<0.001) and increased SEMFs’ migration rate (104cfu/ml: 130.2%, p<0.05). Conclusion This study provides evidence that not only both probiotic strains remain viable in SEMFs’ culture medium but are also able to interact with SEMFs and alter their fibrotic behaviour. Moreover, it was shown that L. plantarum may promote mucosal healing, while S. boulardii could reduce profibrotic responses. Further preclinical research is needed to identify the optimal dosages and combinations of probiotic strains as potential auxiliary treatment of IBD. The study was supported by «Strategic expansion of the Greek Biobanking Infrastructure» (BBMRI-GR) in the framework of the Action “ENHANCING RESEARCH AND INNOVATION INFRASTRUCTURE - SECOND CYCLE” (NSRF 2014–2020) and “AMKE KLEON TSETIS” Foundation, Athens, Greece.

Effects of quercetin on renal autophagy and interstitial fibrosis in diabetes mellitus

To observe the effect of quercetin (Que) on renal autophagy level and renal interstitial fibrosis in the mice with diabetes mellitus (DM),and to discuss the possible mechanism. The wild-type C57BL/6 mice were randomly divided into 3 groups,including normal control (Normal) group, DM group and Que group (n = 9 in every groups). The diabetic mouse model was established by injection of streptozotocin. After 8 weeks of successful replication of the diabetic model,Que was given to the mice in Que group by continuous gavage for 12 weeks,and then the mice in each group were sacrificed to detect the relevant biochemical parameters. The pathological changes of the kidney tissues were observed by HE staining and Masson staining. The levels of the proteins related to autophagy,epithelial-mesenchymal transition(EMT)and fibrosis were determined by Western blot. The mRNA expression of collagen type IV (Col IV),α-smooth muscle actin(α-SMA)and E-cadherin were detected by real-time PCR. Compared with Normal group, fasting blood glucose (FBG), kidney index (KI),serum creatinine, 24-hour urinary albumin excretion rate and 24-hour urine total protein were remarkably increased in DM group (P < 0. 001). The results of HE and Masson staining indicated that renal tissue presented fibrosis in DM group. The protein levels of E-cadherin,beclin-1,microtubule-associated protein 1 light chain 3-II (LC3-II)/LC 3I ratio were reduced in DM group,while the levels of α-SMA,Col IV and Snail1 were increased (P < 0.001). After intervention with Que for 12 weeks,all the relevant biochemical parameters and KI were reduced (P < 0.001) except FBG (P > 0. 05), and renal fibrosis lesions were obviously alleviated. Compared with DM group,the protein levels of E-cadherin,beclin-1 and LC3-II were increased in Res group,but the protein expression levels of α-SMA,Col IV,Snail1 were reduced(P < 0. 001). Compared with DM group, the mRNA level of E-cadherin was increased in Que group, but the mRNA levels of Col IV and α-SMA were reduced (P < 0.001). Quercetin significantly inhibits EMT and reduces renal interstitial fibrosis in diabetic mice,and its mechanism may be related to the promotion of renal autophagy.

Effects and mechanism of hepatocyte growth factor-modified human adipose mesenchymal stem cells on wound healing of full-thickness skin defects in diabetic rats

Objective: To investigate the effects and mechanism of hepatocyte growth factor (HGF)-modified human adipose mesenchymal stem cells (ADSCs) on the wound healing of full-thickness skin defects in diabetic rats. Methods: The experimental research method was adopted. The discarded abdominal adipose tissue was collected from a 35-year-old healthy female who underwent abdominal liposuction in the Department of Plastic Surgery of the First Affiliated Hospital of Air Force Medical University in December 2019. The long spindle-shaped primary ADSCs were obtained by collagenase digestion, and the third passage of cells were identified by flow cytometry to positively express ADSCs surface markers CD29 and CD90 and negatively express CD34 and CD45. The third passage of ADSCs were used for the subsequent experiments. ADSCs were transfected with lentivirus-mediated HGF for 4 h (obtaining HGF modified ADSCs) and then routinely cultured for 24 h. The cell morphology was observed under an inverted phase contrast microscope, and the transfection rate was calculated. Eighty-one male Sprague-Dawley rats aged 4 weeks were induced into diabetic rat model by high glucose and high fat diet combined with streptozotocin injection. A full-thickness skin defect wound of 1.5 cm×1.5 cm was made on the back of each rat. The injured rats were divided into phosphate buffer solution (PBS) group, ADSCs alone group, and HGF-modified ADSCs group according to the random number table, with 27 rats in each group. The rats were injected with the same volume of corresponding substances around the wound on post injury day (PID) 1, 3, and 7, respectively. Nine rats in each group were selected according to the random number table, the wound area of whom was measured on PID 0 (immediately), 3, 7, 10, and 14 (after injection on injection day), and the wound healing rates on PID 3, 7, 10, and 14 were calculated. Nine remaining rats in each group were sacrificed after injection on PID 3 and 7, respectively, and the skin tissue around the wound were collected. The mRNA expressions of inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-10 on PID 3 and collagen type Ⅰ and Ⅲ on PID 7 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The expression level of vascular endothelial growth factor (VEGF) was detected by enzyme-linked immunosorbent assay on PID 7. The protein expression of nuclear factor κb-p65 on PID 3 and phosphorylation level of protein kinase B (Akt) on PID 7 were detected by Western blotting. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: After 24 h of culture, the HGF-transfected human ADSCs showed good morphology, which was not different with the non-transfected ADSCs, and the transfection rate reached 90%. On PID 3, 7, 10, and 14, the wound healing rates of rats in HGF-modified ADSCs group were (31.5±1.0)%, (75.2±2.0)%, (92.2±1.3)%, and (99.1±1.8)%, respectively, being significantly higher than (21.4±1.3)%, (61.4±1.5)%, (80.1±2.1)%, and (92.4±1.8)% in PBS group and (25.1±2.1)%, (67.2±1.3)%, (89.3±1.4)%, and (95.1±2.1)% in ADSCs alone group (t=1.452, 0.393, 0.436, 0.211, 4.982, 3.011, 4.211, 7.503, P<0.05 or P<0.01). On PID 3, compared with those in PBS group and ADSCs alone group, the mRNA expressions of TNF-α and IL-1β and protein expression of nuclear factor κb-p65 in the skin tissue around the wound of rats in HGF-modified ADSCs group were significantly decreased (t=7.281, 17.700, 9.447, 6.231, 13.083, 7.783, P<0.01), and the mRNA expression of IL-10 in the skin tissue around the wound of rats in HGF-modified ADSCs group was significantly increased (t=-6.644, -6.381, P<0.01). On PID 7, compared with those in PBS group and ADSCs alone group, the mRNA expressions of collagen type Ⅰ and Ⅲ, the expression level of VEGF, and the phosphorylation level of Akt in the skin tissue around the wound of rats in HGF-modified ADSCs group were significantly increased (t=-5.126, -4.347, -5.058, -3.367, -10.694, -19.876, -4.890, -6.819, P<0.05 or P<0.01). Conclusions: HGF-modified human ADSCs can significantly promote the wound healing of full-thickness skin defects in diabetic rats. The mechanism may be related to the inhibition of TNF-α and IL-1β expression, the promotion of IL-10, collagen type Ⅰ and Ⅲ, and VEGF expression, which could be related to the inhibition of nuclear factor κB signaling pathway, and the promotion of Akt signaling pathway.

Tonsil-derived mesenchymal stem cells enhance allogeneic bone marrow engraftment via collagen IV degradation

BackgroundCo-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified.MethodsMice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration.ResultsMice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro.ConclusionsCollectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

P067 The effect of a probiotic mix on mucosal healing and fibrotic responses in healthy colonic subepithelial myofibroblasts

Abstract Background Various probiotic strains play a positive role in Inflammatory Bowel Disease and might support mucosal healing. Colonic Subepithelial Myofibroblasts (SEMFs) have a pivotal role in mucosal healing and fibrosis, by migrating to the trauma region and secreting collagen. Our study aimed to investigate whether different strains of probiotics can affect the migration capacity and the fibrotic phenotype of SEMFs isolated from healthy individuals. Methods The probiotic mix of Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium lactis and Saccharomyces boulardii was reconstituted in SEMFs culture medium, they were identified by Gram staining and their viability was assessed by Trypan Blue staining. Primary SEMFs were isolated from colonic biopsies from healthy individuals, and stimulated with 102, 104 or 108 cfu/ml of the mix for 6h or 48h; in 6h total RNA was collected and mRNA expression of collagen type I, III, fibronectin, tissue factor (TF) and α-SMA was measured by quantitative PCR, and in 48h their effect on migration rate through the Wound Healing Assay and collagen secretion via Sircol was assessed. Results Trypan Blue confirmed that the majority of the probiotic mix was alive, and all three bacterial strains and the yeast were stained Gram positive. Stimulation of SEMFs with the probiotic mix resulted in different effects depending on its concentration; 102 cfu/ml downregulated protein collagen secretion (3.4μg/ml, IQR: 2.7–5.0, p&amp;lt;0.01 vs control 6.3μg/ml, IQR: 5.2–6.7) and α-SMA mRNA production (0.7-fold, IQR: 0.7–0.8, p&amp;lt;0.01), 104 cfu/ml upregulated the Fibronectin mRNA expression (1.6-fold, IQR: 1.2–1.8, p&amp;lt;0.0001) and 108 cfu/ml downregulated collagen type I (0.2-fold, IQR: 0.1–0.8, p&amp;lt;0.01) and III mRNAs (0.3-fold, IQR: 0.1–0.8, p&amp;lt;0.01). In addition, all three concentrations resulted in the overall upregulation of TF mRNA expression. Regarding the effect of the probiotic mix on SEMFs migration capacity, 104 cfu/ml of the probiotic mix resulted in increased migration rate by 133.8% (IQR: 102.8–150.2, p&amp;lt;0.01) after 24h, while the use of 102 cfu/ml and 108 cfu/ml decreased it by 58.2% (IQR: 34.8–79.7, p&amp;lt;0.01) and by 46.8% (IQR: 34.3–72.1, p&amp;lt;0.01) respectively. Conclusion This study provides evidence that the probiotic strains L. plantarum, L. acidophillus, B. lactis and S. boulardii remain viable in SEMFs’ culture medium and have the ability to interact with and alter the behavior of SEMFs. Moreover, the effect of the probiotic mix on SEMFs fibrotic phenotype is dependent of the concentration of the probiotics used. Further preclinical research is needed in order to identify the effect of each strain, as well as the suitable dose and combinations in various pathological conditions.

HUMAN AMNION EPITHELIAL CELLS REDUCE BLOOD PRESSURE AND PREVENT AORTIC INFLAMMATION AND COLLAGEN EXPRESSION DURING EXPERIMENTAL HYPERTENSION

Objective: Vascular inflammation and fibrosis are hallmarks of hypertension and contribute to the development of cardiovascular disease. Current treatments for hypertension may reduce blood pressure but do not target the associated pathological changes and end-organ damage. Cell therapy has great therapeutic potential for many conditions as, unlike single pharmacological agents, cells can deliver multiple mediators which could more effectively target complex disease mechanisms. Human amnion epithelial cells (hAECs) have many properties (eg. anti-inflammatory, anti-fibrotic, regenerative and immunologically inert) that make them attractive candidates for a cell-based therapy for vascular pathology. This study aimed to test the potential of hAECs to treat vascular pathology in angiotensin II-induced hypertension. Design and method: Male C57Bl6 mice (8–12 weeks) were administered vehicle (saline; n = 35) or angiotensin II (0.7 mg/kg/d, n = 37) for 14 d via a subcutaneous osmotic minipump. After minipump implantation, a subset of mice were injected with 10^6 of hAECs intravenously. Systolic blood pressure was measured using tail-cuff; inflammation was assessed using flow cytometry and markers of fibrosis using quantitative PCR. Results: Angiotensin II infusion increased systolic blood pressure and promoted accumulation of aortic leukocytes, specifically macrophages and monocytes, which were elevated by ∼3-fold compared to vehicle-infused mice (n = 9–11, P &lt; 0.05). Angiotensin II also increased aortic mRNA expression of collagen type 1 alpha 1 (Col1a1) by ∼4-fold and collagen type 5 alpha 1 (Col5a1) by ∼7-fold compared to vehicle (n = 6–8, P &lt; 0.05). Co-administration of hAECs limited the development of hypertension by angiotensin II (185 ± 5 mmHg vs 165 ± 4 mmHg; n = 9–11, P &lt; 0.05), as well as the aortic infiltration of macrophages and monocytes (n = 9–11, P &lt; 0.05) and expression of Col1a1 and Col5a1 (n = 6–8, P &lt; 0.05). Conclusions: Intravenous administration of hAECs blunted angiotensin II-induced hypertension, aortic inflammation and collagen expression in male mice. This study suggests that hAECs or their cellular products could be explored as treatments for vascular pathology during hypertension.

Pheophorbide a Derivatives Exert Antiwrinkle Effects on UVB-Induced Skin Aging in Human Fibroblasts.

Pheophorbide a is a chlorophyll metabolic breakdown product. This study investigated the antiwrinkle effect of pheophorbide a (PA) and its derivatives, including pyropheophorbide a (PyroPA) and pyropheophorbide a methyl ester (PyroPA-ME), on ultraviolet (UV) B-stimulated CCD-986sk fibroblasts. PA, PyroPA, and PyroPA-ME effectively suppressed reactive oxygen species accumulation in UVB-exposed CCD-986sk fibroblasts. All three pheophorbides also reduced UVB-induced matrix metalloproteinase (MMP)-1 secretion and mRNA expression of MMP-1, MMP-2, and MMP-9. Treatment with pheophorbides resulted in increased procollagen synthesis, and this required enhancement of procollagen type I C-peptide content and mRNA expression of collagen type I alpha 1 (COL1A1) and COL1A2 in CCD-986sk cells. These antiwrinkle effects were more potent with PA and PyroPA than with PyroPA-ME. Furthermore, PA and PyroPA suppressed UVB-induced phosphorylation of extracellular signal-regulated protein kinase and c-Jun N-terminal kinase but not p38. Moreover, all three pheophorbides inhibited NF-κB p65 phosphorylation. Therefore, these pheophorbides, especially PA and PyroPA, can be used as antiwrinkle agents, and PA- or PyroPA-rich natural resources can be used in functional cosmetics.

14 Characterization of adipose tissue extracellular matrix component mRNA expression during porcine adipogenesis using real-time PCR

Abstract Given adipose tissue is histologically classified as connective tissue, we hypothesized expression of extracellular matrix (ECM) components are significantly altered during adipogenesis. However, little is known about the regulation of the ECM during adipose tissue development in the pig. Therefore, the objective of this study was to characterize expression of ECM components during porcine adipogenesis. Primary cultures of adipose tissue stromal-vascular cells were harvested from 3-day-old neonatal pigs (n=6) and preadipocytes induced to differentiate in vitro for 8 days in the presence of insulin, hydrocortisone, and rosiglitazone. Total RNA was extracted from these cultures on days 0 and 8 post-induction. Real-time PCR was then utilized to determine changes in mRNA expression for collagen type I alpha 1 chain (COL1A), collagen type I alpha 2 chain (COL2A), collagen type I alpha 3 chain (COL3A), collagen type I alpha 4 chain (COL4A), collagen type I alpha 6 chain (COL6A), biglycan, fibronectin, laminin, nitogen-1 (NID1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 9 (MMP9), metallopeptidase inhibitor 3 (TIMP3). The mRNA abundances of COL1A, COL3A and MMP2 were significantly downregulated 2.86-fold (P &amp;lt; 0.05), 16.7-fold (P &amp;lt; 0.01) and 3.1-fold (P &amp;lt; 0.05) respectively in day 8 (differentiated) compared to day 0 (undifferentiated) cultures. Meanwhile, mRNA abundances were significantly upregulated during adipogenesis for the COL2A (2.82-fold; P &amp;lt; 0.05), COL4A (2.01-fold; P &amp;lt; 0.05), COL6A (2.8-fold; P &amp;lt; 0.05), biglycan (49.9- fold; P &amp;lt; 0.001), fibronectin (452-fold; P &amp;lt; 0.001), laminin (6.1-fold; P &amp;lt; 0.05), NID1(47.4-fold; P &amp;lt; 0.01), MMP9 (76.8- fold; P &amp;lt; 0.01), and TIMP3(3.04-fold; P &amp;lt; 0.05) genes. These data support the hypothesis that significant changes in ECM components occur during porcine adipogenesis. Modulating adipose tissue ECM remodeling might be a novel strategy to manipulate adiposity in the pig.