The effect of external fixator on bone metabolism in patients with open fracture of extremities.

This experiment aimed to explore the influence mechanism of external fixator on open fracture. A total of 128 patients with open tibiofibular fractures were included in this study. The patients were randomly divided into external fixator group (n=64) and control group (n=64) according to the order of admission. Double-blind controlled observation was used. The levels of osteocalcin (BGP), ß-CTX, P1 NP, BALP, including haptoglobin (Hp), ceruloplasmin (CER), serum adrenocorticotropic hormone (ACTH), cortisol (COR), C-reactive protein (CRP), white blood cell (WBC) and interleukin-6 (IL-6) were recorded in different groups. The postoperative VAS score and quality of life were recorded. Log-rank was used to analyze the difference in postoperative adverse reaction rates among different groups. External fixation stent treatment increased BGP, PINP, and BALP expression and decreased ß-CTX, Hp, CER, ACTH, COR, CRP, WBC, and IL-6 levels. Patients in the external fixation stent group had significantly lower VAS score quality of life scores and incidence of adverse events than the control group. External fixation stents protect open fracture patients by promoting bone metabolism.

Ginsenoside Rc alleviates osteoporosis by the TGF-ß/Smad signaling pathway.

Osteoporosis is a common chronic bone disorder in postmenopausal women. Ginsenosides are primary active components in ginseng and the effects of various ginsenoside variants in osteoporosis treatment have been widely revealed. We planned to explore the impact of ginsenoside Rc on bone resorption in an osteoporosis rat model. We used ovariectomized rats to assess the potential impact of ginsenoside Rc on osteoporosis. µ-CT was implemented for analyzing the microstructure of the distal left femur in rats. H&E staining together with Masson staining were applied for bone histomorphometry evaluation. ELISA kits were implemented to detect serum concentrations of TRACP-5b, OCN, CTX, as well as PINP. Ginsenoside Rc treatment lessened the serum levels of TRACP-5b as well as CTX, while increasing serum levels of OCN, and PINP of OVX rats. Moreover, we found that ginsenoside Rc contributed to the synthesis of type I collagen via increasing Col1a1 and Col1a2 levels in femur tissues of ovariectomized rats. Our findings also revealed that ginsenoside Rc activated the TGF-ß/Smad pathway by increasing TGF-ß as well as phosphorylated Smad2/3 protein levels. Ginsenoside Rc alleviates osteoporosis in rats through promoting the TGF-ß/Smad pathway.

A model for collagen secretion by intercompartmental continuities.

Newly synthesized secretory proteins are exported from the endoplasmic reticulum (ER) at specialized subcompartments called exit sites (ERES). Cargoes like procollagen are too large for export by the standard COPII-coated vesicle of 60 nm average diameter. We have previously suggested that procollagen is transported from the ER to the next secretory organelle, the ER-Golgi intermediate compartment (ERGIC), in TANGO1-dependent interorganelle tunnels. In the theoretical model presented here, we suggest that intrinsically disordered domains of TANGO1 in the ER lumen induce an entropic contraction, which exerts a force that draws procollagen toward the ERES. Within this framework, molecular gradients of pH and/or HSP47 between the ER and ERGIC create a force in the order of tens of femto-Newtons. This force is substantial enough to propel procollagen from the ER at a speed of approximately 1 nm · s-1. This calculated speed and the quantities of collagen secreted are similar to its observed physiological secretion rate in fibroblasts, consistent with the proposal that ER export is the rate-limiting step for procollagen secretion. Hence, the mechanism we propose is theoretically adequate to explain how cells can utilize molecular gradients and export procollagens at a rate commensurate with physiological needs.

Uterine fibroid cell cytoskeletal organization is affected by altered G protein-coupled estrogen receptor-1 and phosphatidylinositol 3-kinase signaling.

OBJECTIVE: To determine whether cyclic strain affects fibroid cell cytoskeletal organization, proliferation, and collagen synthesis differently than myometrial cells. DESIGN: A basic science study using primary cultures of patient-matched myometrial and fibroid cells. SETTING: Academic laboratory. PATIENT(S): Premenopausal women undergoing myomectomy or hysterectomy for the treatment of symptomatic uterine fibroids. INTERVENTION(S): Application of uniaxial strain patterns mimicking periovulation, menses, or dysmenorrhea using the Flexcell tension system or static control. Secondarily, inhibition of G protein-coupled estrogen receptor-1 and phosphatidylinositol 3-kinase. MAIN OUTCOME MEASURE(S): Cell alignment, cell number, and collagen content. RESULT(S): Menses-strained cells demonstrated the most variation in cell alignment, cell proliferation, and procollagen content between myometrial and fibroid cells. Procollagen content decreased in myometrial cells with increasing strain amplitude and decreasing frequency. G protein-coupled estrogen receptor-1 inhibition decreases cellular alignment in the presence of strain. CONCLUSION(S): Mechanotransduction affecting cytoskeletal arrangement through the G protein-coupled estrogen receptor-1-phosphatidylinositol 3-kinase pathway is altered in fibroid cells. These results highlight the importance of incorporating mechanical stimulation into the in vitro study of fibroid pathology.

Phytohormones Affect Differentiation Status of Human Skin Fibroblasts via UPR Activation.

Normalization of secretory activity and differentiation status of mesenchymal cells, including fibroblasts, is an important biomedical problem. One of the possible solutions is modulation of unfolded protein response (UPR) activated during fibroblast differentiation. Here, we investigated the effect of phytohormones on the secretory activity and differentiation of cultured human skin fibroblasts. Based on the analysis of expression of genes encoding UPR markers, abscisic acid (ABA) upregulated expression of the GRP78 and ATF4 genes, while gibberellic acid (GA) upregulated expression of CHOP. Evaluation of the biosynthetic activity of fibroblasts showed that ABA promoted secretion and synthesis of procollagen I and synthesis of fibronectin, as well as the total production of collagen and non-collagen proteins of the extracellular matrix (ECM). ABA also stimulated the synthesis of smooth muscle actin α (α-SMA), which is the marker of myofibroblasts, and increased the number of myofibroblasts in the cell population. On the contrary, GA increased the level of fibronectin secretion, but reduced procollagen I synthesis and the total production of the ECM collagen proteins. GA downregulated the synthesis of α-SMA and decreased the number of myofibroblasts in the cell population. Our results suggest that phytohormones modulate the biosynthetic activity of fibroblasts and affect their differentiation status.

[Sinomenine ameliorates bleomycin A5-induced pulmonary fibrosis by blocking the miR-21/ADAMTS-1 signaling pathway in rats].

Objective To explore the impact of sinomenine on bleomycin A5-induced pulmonary fibrosis (PF) in rats and the underlying mechanism. Methods MRC-5 cells were cultured and treated with sinomenine to determine its optimal concentration and time through the MTT assay. Subsequently, MRC-5 cells were incubated with 80 µmol/L sinomenine for 48 hours or transfected with miR-21 mimic/a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1) siRNA prior to sinomenine treatment. The expression of miR-21, ADAMTS-1, collagen type 1 (Col1) and collagen type 3 (Col3) was detected by quantitative real-time PCR (qRT-PCR) and/or Western blot analysis. Thirty SD rats were randomly divided into control group, sinomenine group and sinomenine combined with miR-21 agomir group, with 10 animals in each group. Bleomycin A5 were intratracheally administered to establish the PF model. Then, rats in control group, sinomenine group and sinomenine +miR-21 agomir group were treated with 9 g/L sodium chloride solution, sinomenine and sinomenine+miR-21 agomir, respectively. On day 28, all rats were sacrificed. HE and Masson staining was performed in pulmonary tissue. The expression of ADAMTS-1, Col1 and Col3 in pulmonary tissue were detected by qRT-PCR and/or Western blot analysis. ELISA was used to measure serum procollagen type 1 carboxyterminal propeptide (P1CP) and procollagen type 3 aminoterminal propeptide (P3NP) levels. Results Administration of sinomenine decreased miR-21 levels, up-regulated ADAMTS-1 expression, and promoted Col1 and Col3 degradation in MRC-5 cells. Importantly, interfering with the miR-21/ADAMTS-1 signaling pathway partially reversed the promotive effect of sinomenine on Col1 and Col3 degradation. Treatment of SD rats with sinomenine reduced alveolitis and PF scores, decreased serum P1CP and P3NP levels, up-regulated pulmonary ADAMTS-1 expression, and down-regulated Col1 and Col3 expression. However, these effects were reversed by miR-21 agomir. Conclusion Sinomenine promotes Col1 and Col3 degradation and inhibits PF in rats by miR-21/ADAMTS-1 pathway.

Impact of essential amino acid intake, resistance exercise, and aging on the concentration of Achilles peritendinous amino acids and procollagen Iα1 in humans.

Recent studies have shown that consuming amino acid-rich compounds improves tendon collagen content and biomechanical properties. Yet, it is unclear if the consumption of amino acids alters local (peritendinous) amino acid concentrations. If aging or exercise influence local amino acid concentrations in conjunction with an amino acid bolus is also not known. We conducted two studies. In Study 1, young women (n = 7, 25 ± 2 years) completed two identical resistance training sessions with either essential amino acid (EAA) or placebo consumption. In Study 2, an EAA bolus identical to Study 1 was given to younger (n = 7; 27 ± 1 year) and older adults (n = 6; 68 ± 2 years). Microdialysis was used to determine Achilles peritendinous amino acid and pro-collagen Iα1 (a marker of collagen synthesis) concentrations. In Study 1, amino acid consumption increased peritendinous concentrations of all EAA except histidine (p < 0.05). In Study 2, the peritendinous concentration of EAAs except for methionine, histidine, and lysine (p > 0.05) increased with time (p < 0.05). Further, the concentrations of most measured amino acids were greater in older adults (p < 0.05). Pro-collagen Iα1 concentration (p > 0.05) was unaffected by exercise, EAA, or aging (p > 0.05). Our findings demonstrate the following: (1) when not combined with exercise, an oral EAA bolus leads to only modest increases in Achilles peritendinous amino acid concentrations; (2) when combined with resistance exercise, EAA consumption resulted in greater peritendinous amino acid concentrations compared to no exercise; (3) the basal concentrations of most amino acids were greater in older adults, and (4) neither the EAA bolus nor exercise altered peritendinous pro-collagen concentrations.

Development of a rapid, efficient, and reusable magnetic bead-based immunocapture system for recombinant human procollagen type II isolation from yeast fermentation broth.

Recombinant collagen production, especially using yeasts as expression systems, could represent a promising alternative over traditional extractive methods from animal sources, offering controllable, scalable, and high-quality products. Monitoring the efficiency and efficacy of procollagen/collagen expression, especially in the initial fermentation phases, can be difficult and time consuming, as biological matrices necessitate purification and commonly used analytical methods are only partially informative. We propose a straightforward, efficient, and reusable immunocapture system able to specifically isolate human procollagen type II from fermentation broths and to release it in few experimental steps. A recovered sample allows for a detailed characterization providing information on structural identity and integrity, which can strongly support the monitoring of fermentation processes. The immunocapture system relies on the use of protein A-coated magnetic beads which have been functionalized and cross-linked with a human anti-procollagen II antibody (average immobilization yield of 97.7%) to create a stable and reusable support for the specific procollagen fishing. We set up the binding and release conditions ensuring specific and reproducible binding with a synthetic procollagen antigen. The absence of non-specific interaction with the support and binding specificity was demonstrated, and the latter was also confirmed by a peptide mapping epitope study in reversed-phase liquid chromatography high-resolution mass spectrometry (RP-LC-HRMS). The bio-activated support proved to be reusable and stable over 21 days from the initial use. Finally, the system was successfully tested on a raw yeast fermentation sample to provide a proof of concept of the applicability within recombinant collagen production.

Dasatinib Attenuates Fibrosis in Keloids by Decreasing Senescent Cell Burden.

Keloids are skin tumours caused by aberrant growth of dermal fibroblasts. Cellular senescence contributes to aging and various pathological conditions, including cancer, atherosclerosis, and fibrotic diseases. However, the effects of cellular senescence and senolytic drugs on keloids remain largely unknown. This study investigated senescent fibroblasts in keloids and assessed the effects of dasatinib on these cells. Tissues acquired from keloid removal surgery were analysed for senescence-associated ß-galactosidase-positive cells, p16 expression, and the effects of dasatinib treatment on keloids. Keloid tissue was xenotransplanted into mice, and the effect of intralesional dasatinib injection on keloid growth was observed. The results showed that the numbers of ß-galactosidase-positive and p16-expressing cells were higher in the keloids compared with in the controls. Dasatinib induced selective clearance of senescent cells and decreased procollagen expression in cultured keloid fibroblasts. In this xenotransplant keloid mouse model, intralesional injection of dasatinib reduced gross keloid tissue weight and the expression of both procollagen and p16. In addition, dasatinib-treated keloid fibroblasts conditioned medium reduced procollagen and p16 expression in cultured keloid fibroblasts. In conclusion, these results suggest that an increased number of senescent fibroblasts may play an important role in the pathogenesis of keloids. Therefore, dasatinib could be an alternative treatment for patients with keloids.

Characterization of a fold in TANGO1 evolved from SH3 domains for the export of bulky cargos.

Bulky cargos like procollagens, apolipoproteins, and mucins exceed the size of conventional COPII vesicles. During evolution a process emerged in metazoans, predominantly governed by the TANGO1 protein family, that organizes cargo at the exit sites of the endoplasmic reticulum and facilitates export by the formation of tunnel-like connections between the ER and Golgi. Hitherto, cargo-recognition appeared to be mediated by an SH3-like domain. Based on structural and dynamic data as well as interaction studies from NMR spectroscopy and microscale thermophoresis presented here, we show that the luminal cargo-recognition domain of TANGO1 adopts a new functional fold for which we suggest the term MOTH (MIA, Otoraplin, TALI/TANGO1 homology) domain. These MOTH domains, as well as an evolutionary intermediate found in invertebrates, constitute a distinct domain family that emerged from SH3 domains and acquired the ability to bind collagen.

Caspase 1 Enhances Transport and Golgi Organization Protein 1 Expression to Promote Procollagen Export From the Endoplasmic Reticulum in Systemic Sclerosis Contributing to Fibrosis.

OBJECTIVE: Transport and Golgi Organization protein 1 (TANGO1) is a protein that regulates the export of procollagen from the endoplasmic reticulum and has a role in the organization of exit sites for general protein export. What regulates the expression of TANGO1 and the role of TANGO1 in fibrosis is poorly understood and has never been studied in the setting of systemic sclerosis (SSc). We undertook this study to determine the role of TANGO1 in SSc fibrosis. METHODS: SSc (n = 15) and healthy (n = 12) primary fibroblast lung cell lines were investigated for the expression of TANGO1. Histologic analyses for TANGO1 were performed on lung biopsy samples (n = 12 SSc patient samples and n = 8 healthy control samples). RESULTS: SSc fibroblasts showed increased expression of TANGO1 protein in cultured fibroblasts. TANGO1 colocalizes with α-smooth muscle actin (α-SMA)-positive cells in SSc lung tissue and is highly up-regulated in the neointima of SSc vessels. TANGO1 expression was dependent on the inflammasome activation of caspase 1. It was also dependent on signaling from the interleukin-1 (IL-1) and transforming growth factor ß (TGFß) receptors. The decrease in TANGO1 down-regulated export of larger cargos including collagen and laminin. Reduced TANGO1 protein had no effect on smaller molecular weight cargoes; however, the secretion of elastin was significantly reduced. CONCLUSION: TANGO1 is markedly increased in SSc fibroblasts and was found to be elevated in lung tissue in association with α-SMA-positive cells. TANGO1 expression is driven by inflammasome-dependent caspase 1 activation and is mediated by IL-1 and TGFß downstream signaling. These observations suggest that during fibrosis, caspase 1 promotes the up-regulation of TANGO1 and the organization of endoplasmic reticulum exits sites, ultimately contributing to procollagen export and fibrosis.

Second-trimester amniotic fluid proteins changes in subsequent spontaneous preterm birth.

INTRODUCTION: The global sequence of the pathogenesis of preterm labor remains unclear. This study aimed to compare amniotic fluid concentrations of extracellular matrix-related proteins (procollagen, osteopontin and IL-33), and of cytokines (IL-19, IL-6, IL-20, TNFα, TGFß, and IL-1ß) in asymptomatic women with and without subsequent spontaneous preterm delivery. MATERIAL AND METHODS: We used amniotic fluid samples of singleton pregnancy, collected by amniocentesis between 16 and 20 weeks' gestation, without stigmata of infection (i.e., all amniotic fluid samples were tested with broad-range 16 S rDNA PCR to distinguish samples with evidence of past bacterial infection from sterile ones), during a randomized, double-blind, placebo-controlled trial to perform a nested case-control laboratory study. Cases were women with a spontaneous delivery before 37 weeks of gestation (preterm group). Controls were women who gave birth at or after 39 weeks (full term group). Amniotic fluid concentrations of the extracellular matrix-related proteins and cytokines measured by immunoassays were compared for two study groups. CLINICALTRIALS: gov: NCT00718705. RESULTS: Between July 2008 and July 2011, in 12 maternal-fetal medicine centers in France, 166 women with available PCR-negative amniotic fluid samples were retained for the analysis. Concentrations of procollagen, osteopontin, IL-19, IL-6, IL-20, IL-33, TNFα, TGFß, and IL-1ß were compared between the 37 who gave birth preterm and the 129 women with full-term delivery. Amniotic fluid levels of procollagen, osteopontin, IL-19, IL-33, and TNFα were significantly higher in the preterm than the full-term group. IL-6, IL-20, TGFß, and IL-1ß levels did not differ between the groups. CONCLUSIONS: In amniotic fluid 16 S rDNA PCR negative samples obtained during second-trimester amniocentesis, extracellular matrix-related protein concentrations (procollagen, osteopontin and IL-33), together with IL-19 and TNFα, were observed higher at this time in cases of later spontaneous preterm birth.

Oleanolic acid regulates the proliferation and extracellular matrix of keloid fibroblasts by mediating the TGF-ß1/SMAD signaling pathway.

BACKGROUND: Keloid (KD) is a unique pathological fibroproliferative disease that seriously affects the appearance of patients. This study investigated the effect of oleanolic acid (OA) on the proliferation of keloid fibroblasts (KFs) and the expression of extracellular matrix (ECM)-related proteins. METHODS: The proliferation of KFs was evaluated using an MTT assay. The effects of OA on intra- and extracellular levels of fibronectin (FN), procollagen I, matrix metalloproteinase-1 (MMP-1), and α-smooth muscle actin (α-SMA) were evaluated using Western blotting. To simulate the KD microenvironment, TGF-ß1 was added to the serum-free culture medium, and KFs were incubated with TGF-ß1 and OA for 24 h. The intra- and extracellular levels of the ECM-related proteins and the effect of OA on TGF-ß1-induced phosphorylation of the SMAD2 and SMAD3 proteins were evaluated using Western blotting. RESULTS: OA inhibited the proliferation of KFs in a concentration- and time-dependent manner. Furthermore, OA treatment of KFs reduced the intra- and extracellular levels of FN, procollagen I, and α-SMA and increased those of MMP-1. OA also reduced TGF-ß1-induced increases in the intra- and extracellular levels of FN, procollagen I, and α-SMA and increased the levels of the MMP-1 protein. Additionally, OA significantly reduced TGF-ß1-induced phosphorylation of SMAD2 and SMAD3 in KFs. CONCLUSIONS: OA inhibited KF proliferation and reduced ECM deposition through the TGF-ß1/SMAD pathway, which suggests that OA may be an effective drug for the prevention and treatment of KD.

Biophotonic Effects of Low-Level Laser Therapy on Adipose-Derived Stem Cells for Soft Tissue Deficiency.

PURPOSES: The objectives of this study are to use diode lasers for low-level laser therapy (LLLT) and to assess its applicability and effects in adipose-derived stem cell (ADSC) growth processes. METHODS: Studies were conducted on the diode laser with wavelengths of 622.7, 527.1, and 467.3 nm. The mechanism of action of LLL illumination was studied on ADSCs, isolated from human tissue, and then cultured by examining different wavelengths to determine the relevant light parameters for optimal responses. We used enzyme-linked immunosorbent assay and real-time polymerase chain to determine the percentages of fibroblast-mediated procollagen type 1 and matrix metallopeptidase 1 (MMP-1), MMP-2, and MMP-9 production at different wavelengths. The levels of lactate dehydrogenase produced by ADSCs after LLL illumination were assessed as well. Clinical results from 20 patients treated for soft tissue deficiency were collected for assessment of ADSC-assisted lipotransfer. RESULTS: Low-level laser (622.7 nm) illumination on cell cultures in vitro increased ADSCs proliferation, type 1 procollagen expression, collagen production, as well as MMP-1, MMP-2, and MMP-9 relative expression. Statistical analysis demonstrated a significant difference in red light (622.7 nm) versus green light (527.1 nm) and blue light (467.3 nm, P < 0.05). No significant differences were noted between the effects of green and blue lights. In clinical application, all patients attained significant improvement with treatment in the final outcome assessment after 6 months. CONCLUSIONS: Low-level laser illumination may affect ADSCs growth processes and ADSC-assisted lipotransfer for soft tissue deformity, scar treatment, wound healing, and other reconstructive surgery.

Cannabidiol inhibits lung proliferation in monocrotaline-induced pulmonary hypertension in rats.

Cannabidiol (CBD) is a safe and well-tolerated plant-derived drug with anti-proliferative properties. Pulmonary hypertension (PH) is a rapidly progressive and still incurable disease. CBD diminishes monocrotaline (MCT)-induced PH, including reduced right ventricular systolic pressure, pulmonary vascular hypertrophy, and right ventricular remodeling. The aim of our study was to investigate the effect of chronic administration of CBD (10 mg/kg once daily for 21 days) on selected remodeling parameters in the lung of MCT-induced PH rats. In MCT-induced PH, we found an increase in profibrotic parameters, e.g., transforming growth factor ß1 (TGF-ß1), galectin-3 (Gal-3), procollagen I, collagen I, C-propeptide, matrix metalloproteinase 9 (MMP-9) and an increased number of mast cells. In our study, we observed that the TGF-ß1, Gal-3, procollagen I, collagen I, C-propeptide, and mast cell levels in lung tissue were decreased after CBD administration to MCT-treated rats. In summary, CBD treatment has an anti-proliferative effect on MCT-induced PH. Given the beneficial multidirectional effects of CBD on PH, we believe that CBD can be used as an adjuvant PH therapy, but this argument needs to be confirmed by clinical trials.

KLHL12 can form large COPII structures in the absence of CUL3 neddylation.

CUL3-RING ubiquitin ligases (CRL3s) are involved in various cellular processes through different Bric-a-brac, Tramtrack, and Broad-complex (BTB)-domain proteins. KLHL12, a BTB-domain protein, is suggested to play an essential role in the export of large cargo molecules such as procollagen from the endoplasmic reticulum (ER). CRL3KLHL12 monoubiquitylates SEC31, leading to an increase in COPII vesicle dimension. Enlarged COPII vesicles can accommodate procollagen molecules. Thus, CRL3KLHL12 is essential for the assembly of large COPII structures and collagen secretion. CRL3s are activated by CUL3 neddylation. Here, we evaluated the importance of CUL3 neddylation in COPII assembly and collagen secretion. Unexpectedly, the assembly of large COPII-KLHL12 structures persisted and cellular collagen levels decreased on treatment with MLN4924, a potent inhibitor of NEDD8-activating enzyme. When we introduced mutations into KLHL12 at the CUL3 interface, these KLHL12 variants did not interact with neddylated CUL3, but one of them (Mut A) still supported large COPII-KLHL12 structures. Overexpression of wild-type KLHL12, but not Mut A, lowered cellular collagen levels most likely via lysosomal degradation. Our results suggest that CUL3 neddylation is not necessary for the formation of large COPII-KLHL12 structures, but active CRL3KLHL12 contributes to the maintenance of collagen levels in the cell.

Circulating levels of endotrophin and cross-linked type III collagen reflect liver fibrosis in people with HIV.

BACKGROUND AND AIMS: Liver-associated complications still frequently lead to mortality in people with HIV (PWH), even though combined antiretroviral treatment (cART) has significantly improved overall survival. The quantification of circulating collagen fragments released during collagen formation and degradation correlate with the turnover of extracellular matrix (ECM) in liver disease. Here, we analysed the levels of ECM turnover markers PC3X, PRO-C5, and PRO-C6 in PWH and correlated these with hepatic fibrosis and steatosis. METHODS: This monocentre, retrospective study included 141 PWH. Liver stiffness and liver fat content were determined using transient elastography (Fibroscan) with integrated CAP function. Serum levels of formation of cross-linked type III collagen (PC3X), formation of type V collagen (PRO-C5) and formation type VI collagen (PRO-C6), also known as the hormone endotrophin, were measured with ELISA. RESULTS: Twenty-five (17.7%) of 141 PWH had clinical significant fibrosis with liver stiffness ≥ 7.1 kPa, and 62 PWH (44.0%) had steatosis with a CAP value > 238 dB/m. Study participants with fibrosis were older (p = 0.004) and had higher levels of AST (p = 0.037) and lower number of thrombocytes compared to individuals without fibrosis (p = 0.0001). PC3X and PRO-C6 were markedly elevated in PWH with fibrosis. Multivariable cox regression analysis confirmed PC3X as independently associated with hepatic fibrosis. PRO-C5 was significantly elevated in participants with presence of hepatic steatosis. CONCLUSION: Serological levels of cross-linked type III collagen formation and endotrophin were significantly associated with liver fibrosis in PWH receiving cART and thus may be suitable as a non-invasive evaluation of liver fibrosis in HIV disease.

miR-449a ameliorates acute rejection after liver transplantation via targeting procollagen-lysine1,2-oxoglutarate5-dioxygenase 1 in macrophages.

Acute rejection (AR) is an important factor that leads to poor prognosis after liver transplantation (LT). Macrophage M1-polarization is an important mechanism in AR development. MicroRNAs play vital roles in disease regulation; however, their effects on macrophages and AR remain unclear. In this study, rat models of AR were established following LT, and macrophages and peripheral blood mononuclear cells were isolated from rats and humans, respectively. We found miR-449a expression to be significantly reduced in macrophages and peripheral blood mononuclear cells. Overexpression of miR-449a not only inhibited the M1-polarization of macrophages in vitro but also improved the AR of transplant in vivo. The mechanism involved inhibiting the noncanonical nuclear factor-kappaB (NF-κB) pathway. We identified procollagen-lysine1,2-oxoglutarate5-dioxygenase 1 (PLOD1) as a target gene of miR-449a, which could reverse miR-449a's inhibition of macrophage M1-polarization, amelioration of AR, and inhibition of the NF-κB pathway. Overall, miR-449a inhibited the NF-κB pathway in macrophages through PLOD1 and also inhibited the M1-polarization of macrophages, thus attenuating AR after LT. In conclusion, miR-449a and PLOD1 may be new targets for the prevention and mitigation of AR.

Poly-L-Lactic acid increases collagen gene expression and synthesis in cultured dermal fibroblast (Hs68) through the TGF-ß/Smad pathway.

OBJECTIVE: Poly-L-Lactic Acid (PLLA) is a synthetic polymer which possesses biocompatible and biodegradable properties, and is widely used in the clinical filler material. This study focuses on the potential role of PLLA on the collagen production of dermal fibroblasts and its mechanism. METHODS: The dermal fibroblast Hs60 was treated with different concentration of PLLA. RT-qPCR was conducted for the determination of mRNA levels of collagen type I (COL1) alpha 1 (COL1A1), COL1 alpha 2 (COL1A2), elastin, matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2. Procollagen Type I C-peptide (PIP) enzyme immunoassay (EIA) Kit assay was carried out to analyze procollagen production. Western Blot was employed to examine the effect of PLLA and transforming frown factor (TGF-ß) receptor-specific inhibitor (SB431542) on protein levels of COL1A1 and TGF-ß/Smad signaling pathway related proteins. RESULTS: With the increase of PLLA concentration, the production of procollagen gradually increased, and both protein and mRNA levels of COL1A1 and COL1A2 gradually increased (p < 0.001). Elevated PLLA concentrations increased elastin, TIMP-1, and TIMP-2 levels and attenuated MMP-1 expression. PLLA increased TGF-ß levels in a dose-dependently manner. p-Smad2 and p-Smad3 protein levels were also increased by PLLA, but the influences were reversed by SB431542 (p < 0.001). Similarly, increased levels of COL1A1, COL1A2, TIMP-1, and TIMP-2 caused by PLLA were significantly inhibited by SB431542, whereas MMP-1 was typically elevated (p < 0.001). CONCLUSION: Poly-L-Lactic Acid promotes the collagen production of dermal fibroblasts by activating the TGF-ß/Smad signaling pathway. The findings may lay a foundation for clinical material applications of PLLA.

Sinomenine ameliorates bleomycin A5-induced pulmonary fibrosis by blocking the miR-21/ADAMTS-1 signaling pathway in rats / 细胞与分子免疫学杂志

Objective To explore the impact of sinomenine on bleomycin A5-induced pulmonary fibrosis (PF) in rats and the underlying mechanism. Methods MRC-5 cells were cultured and treated with sinomenine to determine its optimal concentration and time through the MTT assay. Subsequently, MRC-5 cells were incubated with 80 μmol/L sinomenine for 48 hours or transfected with miR-21 mimic/a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1) siRNA prior to sinomenine treatment. The expression of miR-21, ADAMTS-1, collagen type 1 (Col1) and collagen type 3 (Col3) was detected by quantitative real-time PCR (qRT-PCR) and/or Western blot analysis. Thirty SD rats were randomly divided into control group, sinomenine group and sinomenine combined with miR-21 agomir group, with 10 animals in each group. Bleomycin A5 were intratracheally administered to establish the PF model. Then, rats in control group, sinomenine group and sinomenine +miR-21 agomir group were treated with 9 g/L sodium chloride solution, sinomenine and sinomenine+miR-21 agomir, respectively. On day 28, all rats were sacrificed. HE and Masson staining was performed in pulmonary tissue. The expression of ADAMTS-1, Col1 and Col3 in pulmonary tissue were detected by qRT-PCR and/or Western blot analysis. ELISA was used to measure serum procollagen type 1 carboxyterminal propeptide (P1CP) and procollagen type 3 aminoterminal propeptide (P3NP) levels. Results Administration of sinomenine decreased miR-21 levels, up-regulated ADAMTS-1 expression, and promoted Col1 and Col3 degradation in MRC-5 cells. Importantly, interfering with the miR-21/ADAMTS-1 signaling pathway partially reversed the promotive effect of sinomenine on Col1 and Col3 degradation. Treatment of SD rats with sinomenine reduced alveolitis and PF scores, decreased serum P1CP and P3NP levels, up-regulated pulmonary ADAMTS-1 expression, and down-regulated Col1 and Col3 expression. However, these effects were reversed by miR-21 agomir. Conclusion Sinomenine promotes Col1 and Col3 degradation and inhibits PF in rats by miR-21/ADAMTS-1 pathway.