Cytotoxic T cells drive doxorubicin-induced cardiac fibrosis and systolic dysfunction.

Doxorubicin, the most prescribed chemotherapeutic drug, causes dose-dependent cardiotoxicity and heart failure. However, our understanding of the immune response elicited by doxorubicin is limited. Here we show that an aberrant CD8+ T cell immune response following doxorubicin-induced cardiac injury drives adverse remodeling and cardiomyopathy. Doxorubicin treatment in non-tumor-bearing mice increased circulating and cardiac IFNγ+CD8+ T cells and activated effector CD8+ T cells in lymphoid tissues. Moreover, doxorubicin promoted cardiac CD8+ T cell infiltration and depletion of CD8+ T cells in doxorubicin-treated mice decreased cardiac fibrosis and improved systolic function. Doxorubicin treatment induced ICAM-1 expression by cardiac fibroblasts resulting in enhanced CD8+ T cell adhesion and transformation, contact-dependent CD8+ degranulation and release of granzyme B. Canine lymphoma patients and human patients with hematopoietic malignancies showed increased circulating CD8+ T cells after doxorubicin treatment. In human cancer patients, T cells expressed IFNγ and CXCR3, and plasma levels of the CXCR3 ligands CXCL9 and CXCL10 correlated with decreased systolic function.

Induction of fibrosis following exposure to bisphenol A and its analogues in 3D human uterine leiomyoma cultures.

Bisphenol A (BPA) and its analogues (BPAF, BPS) are ubiquitous environmental contaminants used as plastic additives in various daily life products, with many concerns on their role as environmental estrogens. Uterine leiomyomas (fibroids) are highly prevalent gynecologic tumors with progressive fibrosis. Fibroids are hormone-responsive and may be the target of environmental estrogens. However, the effects of BPA, BPAF, and BPS exposure on uterine fibrosis are largely unknown. Here, we evaluated fibrosis and the crucial role of TGF-beta signaling in human fibroid tumors, the profibrotic effects of BPA, BPAF or BPS in a human 3D uterine leiomyoma (ht-UtLM) in vitro model, and the long-term outcomes of BPAF exposure in rat uterus. In 3D ht-UtLM spheroids, BPA, BPAF, and BPS all promoted cell proliferation and fibrosis by increasing the production of extracellular matrices. Further mechanistic analysis showed the profibrotic effects were induced by TGF-beta signaling activation mainly through SMAD2/3 pathway and crosstalk with multiple non-SMAD pathways. Furthermore, the profibrotic effects of BPAF were supported by observation of uterine fibrosis in vivo in rats following long-term BPAF exposure. Overall, the 3D ht-UtLM spheroid can be an important model for investigating environment-induced fibrosis in uterine fibroids. BPA and its analogues can induce fibrosis via TGF-beta signaling.

The hippo-YAP1/HIF-1α pathway mediates arsenic-induced renal fibrosis.

Epidemiological evidence reveals that arsenic increases the risk of chronic kidney disease (CKD) in humans, but its mechanism of action has so far been unclear. Fibrosis is the manifestation of end-stage renal disease. Hypoxia is recognized as a vital event accompanying the progression of renal fibrosis. KM mice were exposed to 0, 20, 40, and 80 mg/L NaAsO2 for 12 weeks. HK-2 cells were treated with 1 µM NaAsO2 for 4 weeks. The results showed that arsenic increased the expression of hypoxia-inducible factor 1α (HIF-1α) (P < 0.05), which is involved in inorganic arsenic-induced renal fibrosis. The Hippo signaling pathway is the upstream signal of HIF-1α and the kinase cascade of Large tumor suppressor kinase 1 (LATS1) and Yes-associated protein 1 (YAP1) is the heart of the Hippo pathway. Our results showed that protein expressions of LATS1 and phosphorylated YAP1 were decreased, and dephosphorylated YAP1 expression increased in arsenic-treated mouse kidneys and human HK-2 cells (P < 0.05). Our research manifested that arsenic treatment suppressed the Hippo signaling and induced high expression of YAP1 into the nucleus. We also found that YAP1 was involved in arsenic-induced renal fibrosis by forming a complex with HIF-1α and maintaining HIF-1α stability. Our findings indicate that YAP1 is a potential target for molecular-based therapy for arsenic-mediated renal fibrosis.

Effects of sacubitril-valsartan on remodelling, fibrosis and mitochondria in a murine model of isoproterenol-induced left ventricular dysfunction.

BACKGROUND: Sacubitril/valsartan has been demonstrated to promote left ventricular (LV) reverse remodelling and improve outcomes in patients with heart failure (HF) with reduced ejection fraction (EF). Its molecular and tissue effects have not been fully elucidated yet, due to the paucity of preclinical studies, mostly based on ischaemic models. We aimed to evaluate the effects of sacubitril/valsartan on LV remodelling, myocardial fibrosis and mitochondrial biology in a murine model of non-ischaemic LV dysfunction. METHODS: Adult transgenic male mice with cardiac-specific hyperaldosteronism (AS mice) received subcutaneous isoproterenol injections to induce LV systolic dysfunction. After 7 days, mice were randomized to a 2-week treatment with saline (ISO-AS n = 15), valsartan (ISO + V n = 12) or sacubitril/valsartan (ISO + S/V n = 12). Echocardiography was performed at baseline, at day 7, and after each of the 2 weeks of treatment. After sacrifice at day 21, histological and immunochemical assays were performed. A control group of AS mice was also obtained (Ctrl-AS n = 8). RESULTS: Treatment with sacubitril/valsartan, but not with valsartan, induced a significant improvement in LVEF (p = 0.009 vs ISO-AS) and fractional shortening (p = 0.032 vs ISO-AS) after 2- week treatment. In both ISO + V and ISO + S/V groups, a trend toward reduction of the cardiac collagen 1/3 expression ratio was detected. ISO + V and ISO + S/V groups showed a significant recovery of mitochondrial morphology and inner membrane function meant for oxidative phosphorylation. CONCLUSION: In a murine model of non-ischaemic HF, sacubitril/valsartan proved to have beneficial effects on LV systolic function, and on cardiac energetics, by improving mitochondrial activity.

HIF-1α and adaptor protein LIM and senescent cell antigen-like domains protein 1 axis promotes tubulointerstitial fibrosis by interacting with vimentin in angiotensin II-induced hypertension.

BACKGROUND AND PURPOSE: Activation of the renin-angiotensin system, as a hallmark of hypertension and chronic kidney diseases (CKD) is the key pathophysiological factor contributing to the progression of tubulointerstitial fibrosis. LIM and senescent cell antigen-like domains protein 1 (LIMS1) plays an essential role in controlling of cell behaviour through the formation of complexes with other proteins. Here, the function and regulation of LIMS1 in angiotensin II (Ang II)-induced hypertension and tubulointerstitial fibrosis was investigated. EXPERIMENTAL APPROACH: C57BL/6 mice were treated with Ang II to induce tubulointerstitial fibrosis. Hypoxia-inducible factor-1α (HIF-1α) renal tubular-specific knockout mice or LIMS1 knockdown AAV was used to investigate their effects on Ang II-induced renal interstitial fibrosis. In vitro, HIF-1α or LIMS1 was knocked down or overexpressed in HK2 cells after exposure to Ang II. KEY RESULTS: Increased expression of tubular LIMS1 was observed in human kidney with hypertensive nephropathy and in murine kidney from Ang II-induced hypertension model. Tubular-specific knockdown of LIMS1 ameliorated Ang II-induced tubulointerstitial fibrosis in mice. Furthermore, we demonstrated that LIMS1 was transcriptionally regulated by HIF-1α in tubular cells and that tubular HIF-1α knockout ameliorates LIMS1-mediated tubulointerstitial fibrosis. In addition, LIMS1 promotes Ang II-induced tubulointerstitial fibrosis by interacting with vimentin. CONCLUSION AND IMPLICATIONS: We conclude that HIF-1α transcriptionally regulated LIMS1 plays a central role in Ang II-induced tubulointerstitial fibrosis through interacting with vimentin. Our finding represents a new insight into the mechanism of Ang II-induced tubulointerstitial fibrosis and provides a novel therapeutic target for progression of CKD.

Evaluating the potential of daily intake of polystyrene microplastics via drinking water in inducing PCOS and its ovarian fibrosis progression using female zebrafish.

Polystyrene microplastics, extensively considered endocrine disrupting chemicals, disturb the reproductive system of living organisms. Polycystic ovary syndrome (PCOS), the reproductive endocrinopathy, is longstanding concern due to its eternal impacts as reproductive disorder and infertility. Despite several reports in reproductive and endocrine toxicity, there is inadequate literature regarding the daily intake of polystyrene-microplastics via drinking water in causing PCOS and leading to ovarian fibrosis in long-term. The present study investigated whether daily consumption of polystyrene-microplastics at doses equivalent to human exposure can cause PCOS and progress to ovarian fibrosis, using female zebrafish as model. Resembling letrozole-PCOS zebrafish model, daily intake of polystyrene-microplastics displayed hallmark PCOS pathophysiology; like excess body weight and %Gonadosomatic index, decreased Follicle Stimulating Hormone and ß-estradiol, increased Luteinising Hormone, brain and ovarian Testosterone (39.3% and 75% respectively). Correspondingly, ovarian histology revealed more developing (stage I and II) oocytes and less mature oocytes alongwith cystic lesions; like follicular membrane disorganization, zona pellucida invagination, theca hypertrophy, basophilic granular accumulation and oocyte buddings. Lipid deposition in intestinal and ovarian tissues was evidenced and increased fasting blood glucose manifesting insulin resistance. The expression of PCOS biomarkers (tox3, dennd1a, fem1a) was significantly disturbed. Polystyrene microplastics played vital role in inducing PCOS further enhancing oxidative stress, which positively influences inflammation and aggravate ovarian mitophagy, shedding light on its ability to harshen PCOS into ovarian fibrosis, which is characterized by collagen deposition and upregulation of pro-fibrogenic biomarker genes. These findings illustrate the potential of daily microplastics intake via drinking water in triggering PCOS and its progression to ovarian fibrosis.

Skin and lung fibrosis induced by bleomycin in mice: a systematic review.

OBJECTIVE: Scleroderma, or systemic sclerosis (SSc), is a chronic autoimmune connective disease with an unknown etiology and poorly understood pathogenesis. The striking array of autoimmune, vascular, and fibrotic changes that develop in almost all patients makes SSc unique among connective tissue diseases. Although no animal model developed for SSc to date fully represents all features of human disease, some animal models that demonstrate features of SSc may help to better understand the pathogenesis of the disease and to develop new therapeutic options. In this review, we aimed to evaluate skin fibrosis and lung involvement in a bleomycin (BLM)-induced mouse model and to evaluate the differences between studies. METHODS: A systematic literature review (PRISMA guideline) on PubMed and EMBASE (until May 2023, without limits) was performed. A primary literature search was conducted using the PubMed and EMBASE databases for all articles published from 1990 to May 2023. Review articles, human studies, and non-dermatological studies were excluded. Of the 38 non-duplicated studies, 20 articles were included. RESULTS: Among inducible animal models, the BLM-induced SSc is still the most widely used. In recent years, the measurement of tissue thickness between the epidermal-dermal junction and the dermal-adipose tissue junction (dermal layer) has become more widely accepted. CONCLUSIONS: In animal studies, it is important to simultaneously evaluate lung tissues in addition to skin fibrosis induced in mice by subcutaneous BLM application, following the 3R (replacement, reduction, and refinement) principle to avoid cruelty to animals.

Arsenic exposure induced renal fibrosis via regulation of mitochondrial dynamics and the NLRP3-TGF-ß1/SMAD signaling pathway.

Environmental arsenic exposure is one of the major global public health problems. Studies have shown that arsenic exposure can cause renal fibrosis, but the underlying mechanism is still unclear. Integrating the in vivo and in vitro models, this study investigated the potential molecular pathways for arsenic-induced renal fibrosis. In this study, SD rats were treated with 0, 5, 25, 50, and 100 mg/L NaAsO2 for 8 weeks via drinking water, and HK2 cells were treated with different doses of NaAsO2 for 48 h. The in vivo results showed that arsenic content in the rats' kidneys increased as the dose increased. Body weight decreased and kidney coefficient increased at 100 mg/L. As a response to the elevated NaAsO2 dose, inflammatory cell infiltration, renal tubular injury, glomerular atrophy, tubulointerstitial hemorrhage, and fibrosis became more obvious indicated by HE and Masson staining. The kidney transcriptome profiles further supported the protein-protein interactions involved in NaAsO2-induced renal fibrosis. The in vivo results, in together with the in vitro experiments, have revealed that exposure to NaAsO2 disturbed mitochondrial dynamics, promoted mitophagy, activated inflammation and the TGF-ß1/SMAD signaling pathway, and finally resulted in fibrosis. In summary, arsenic exposure contributed to renal fibrosis via regulating the mitochondrial dynamics and the NLRP3-TGF-ß1/SMAD signaling axis. This study presented an adverse outcome pathway for the development of renal fibrosis due to arsenic exposure through drinking water.

Nrf2 protects against renal fibrosis induced by chronic cadmium exposure in mice.

Environmental cadmium (Cd) exposure is a serious public health concern, as the kidney is the primary target for Cd exposure. The present study aimed to investigate the role and underlying mechanisms of nuclear factor erythroid-derived 2-like 2 (Nrf2) in renal fibrosis induced by chronic Cd exposure. Nrf2 knockout (Nrf2-KO) mice and their wild-type littermates (Nrf2-WT) were exposed to 100 or 200 ppm Cd in drinking water for up to 16 or 24 weeks. Following the Cd exposures, Nrf2-KO mice showed elevated urinary neutrophil gelatinase-associated lipocalin (NGAL) and BUN levels compared to Nrf2-WT mice. Masson's trichrome staining and expression of fibrosis-associated proteins revealed that more severe renal fibrosis occurred in Nrf2-KO than that in Nrf2-WT mice. Renal Cd content in the Nrf2-KO mice exposed to 200 ppm Cd was lower than that in Nrf2-WT mice, which might be a consequence of the severe renal fibrosis in the Nrf2-KO mice. Mechanistic studies showed that Nrf2-KO mice exhibited higher levels of oxidative damage, lower antioxidant levels, and more regulated cell death, apoptosis in particular, than those in Nrf2-WT mice caused by Cd exposure. In conclusion, Nrf2-KO mice were more prone to develop renal fibrosis induced by chronic Cd exposure, partially due to a weakened antioxidant, detoxification capacity and increased oxidative damage.

Prolonged exposure to the herbicide atrazine promotes kidney fibrosis by activating Wnt/ß-catenin signaling in rats.

Exposure to atrazine (ATR), a widely-used herbicide, is a potential harmful to human health due to its long-term environmental persistence and bioaccumulation. The effects of chronic exposure to ATR on renal function in rats were evaluated in this research. Female Sprague-Dawley rats at 4 weeks of age were treated with different concentrations of ATR for 6 months. No significant differences  in terms of renal functions were observed after ATR treatment. In histopathological examination of the kidney, Hematoxylin-Eosin staining indicated the development of degenerative changes in a dose-dependent manner. The results revealed that ATR exposure leads to renal fibrosis and that activation of the Wnt/ß-catenin pathway plays a potential role in ATR-related renal fibrosis. Levels of transforming growth factor (TGF)-ß and TGF-ß1 levels and the reactive oxygen species were significantly upregulated after ATR treatment. In conclusion, long-term exposure to ATR could cause kidney fibrosis, which is the result of epithelial-mesenchymal transition caused by inflammation and oxidative stress.

Protective effects of alpha-lipoic acid on bleomycin-induced skin fibrosis through the repression of NADPH Oxidase 4 and TGF-ß1/Smad3 signaling pathways.

The aim of this study was to determine the protective effects of alpha-lipoic acid (ALA), which is known as a powerful antioxidant, and the possible related molecular mechanisms that mediate its favorable action on skin fibrosis in the bleomycin (BLM)-induced scleroderma (SSc) model in mice. The experimental design was established with four groups of eight mice: Control, ALA (100 mg/kg), BLM (5 µg/kg), and BLM + ALA group. BLM was administered via subcutaneous (sc) once a day while ALA was injected intraperitoneally (ip) twice a week for 21 days. Histopathological and biochemical analyses showed that ALA significantly reduced BLM-induced dermal thickness, inflammation score, and mRNA expression of tumor necrosis factor-alpha (TNF-α) in the skin. Besides, the mRNA expressions of the subunits of NADPH oxidase, which are Nox4 and p22phox, were found to be significantly induced in the BLM group. However, ALA significantly reduced their mRNA expression, which were in parallel to its decreasing effect on serum total oxidant status (TOS) level. Moreover, it was found that ALA downregulated the mRNA expressions of alpha-smooth muscle actin (α-SMA), collagen type I and fibronectin in the skin tissue of the BLM group. Additionally, it was shown that ALA reduced significantly the TGF-ß1 and p-Smad3 protein expressions in the BLM + ALA group. On the other hand, ALA did not exhibit any significant effect on the p38 mitogen-activated kinase (MAPK) activation induced by BLM. All these findings point out that ALA may be a promising treatment for the attenuation of skin fibrosis in SSc patients.

Hepatoprotective effect of Baicalin against thioacetamide-induced cirrhosis in rats: Targeting NOX4/NF-κB/NLRP3 inflammasome signaling pathways.

AIM: Liver cirrhosis is the result of a vicious cycle of both chronic oxidative stress and inflammation. NADPH oxidase-4 (NOX4) and its companion, NOD-like receptor protein 3 (NLRP3) inflammasome, are emerging as therapeutic targets of liver fibrosis. MAIN METHODS: Baicalin (BA), a natural flavone, has been investigated for its therapeutic potential against cirrhosis induced by thioacetamide (TAA) (200 mg/kg, twice/week) for 12 weeks in Sprague-Dawley rats. Two doses of BA were administered (25 and 75 mg/kg/day, orally, a week after TAA was stopped and continued for 4 weeks). KEY FINDINGS: BA was able to reduce fibrosis visualized by Masson trichrome and immunohistochemical staining of the hepatic α-smooth muscle actin (α-SMA) and transforming growth factor-ß1. Moreover, BA was able to ameliorate inflammation by reducing hepatic NLRP3 inflammasome subunits, NLRP3 and caspase-1, both parts of the complex responsible for the activation of different interleukins (IL), measured as IL-1ß. In addition, BA was able to reduce hepatic nuclear factor kappa B (NF-κB)-driven inflammation through IL-6. BA targeted inflammation through its anti-oxidant ability evidenced by the enhancement of the hepatic superoxide dismutase (SOD) and reduced glutathione (GSH) activity and level, respectively, and the reduction of both hepatic malondialdehyde (MDA) and nitric oxide (NOx) contents. Treatment with BA significantly decreased TAA-induced elevation in hepatic NOX4, a key enzyme for reactive oxygen species (ROS) generation, as well as, inducible nitric oxide synthase (iNOS). SIGNIFICANCE: therefore, the study could conclude, the anti-fibrotic effect of BA through TGF- ß1/NOX4/NF-κB/NLRP3 pathway, exerting both anti-inflammatory and anti-oxidant effects.

Adefovir accumulation in the renal interstitium triggers mast cell degranulation and promotes renal interstitial fibrosis.

Organic anion transporters 1 (OAT1) and OAT3 are responsible for transporting adefovir (ADV) into renal tubular epithelial cells. Our previous research found that ADV accumulated in the renal interstitium and caused renal interstitial fibrosis when Oat1/3 were inhibited by OATs inhibitor probenecid for long-term. Mast cells (MCs) in the interstitial space are considered to be key drivers of renal fibrosis. The current work investigated the effect of ADV on MCs in vitro and during the development of interstitial fibrosis in rats. Results indicate that ADV triggers chymase release from cultured RBL-2H3 mast cells in a time-and concentration-dependent manner. Angiotensin II (Ang II) in renal interstitium is generated mainly by chymase, renin and other products released from MCs, and has a direct effect on fibrosis through the angiotensin receptor. The concentrations of Ang II and fibrosis was significantly increased after administration of ADV alone or with probenecid for 4 weeks. The MCs membrane stabilizer sodium cromoglycate (SCG) and the angiotensin receptor antagonist Valsartan (VAL) could ameliorate ADV-induced nephrotoxicity. Additionally, SCG or VAL could reduce the accumulation of ADV in the renal interstitium by upregulating the expression of Oat1/3 and multidrug resistance-associated protein 4. Therefore, ADV accumulation in the renal interstitium could promote the degranulation of interstitial MCs and drive the development of renal fibrosis. SCG or VAL could ameliorate ADV-associated fibrosis by decreasing degranulation of MCs and accelerating renal clearance of ADV.

Short-term tetrabromobisphenol A exposure promotes fibrosis of human uterine fibroid cells in a 3D culture system through TGF-beta signaling.

Tetrabromobisphenol A (TBBPA), a derivative of BPA, is a ubiquitous environmental contaminant with weak estrogenic properties. In women, uterine fibroids are highly prevalent estrogen-responsive tumors often with excessive accumulation of extracellular matrix (ECM) and may be the target of environmental estrogens. We have found that BPA has profibrotic effects in vitro, in addition to previous reports of the in vivo fibrotic effects of BPA in mouse uterus. However, the role of TBBPA in fibrosis is unclear. To investigate the effects of TBBPA on uterine fibrosis, we developed a 3D human uterine leiomyoma (ht-UtLM) spheroid culture model. Cell proliferation was evaluated in 3D ht-UtLM spheroids following TBBPA (10-6 -200 µM) administration at 48 h. Fibrosis was assessed using a Masson's Trichrome stain and light microscopy at 7 days of TBBPA (10-3  µM) treatment. Differential expression of ECM and fibrosis genes were determined using RT² Profiler™ PCR arrays. Network and pathway analyses were conducted using Ingenuity Pathway Analysis. The activation of pathway proteins was analyzed by a transforming growth factor-beta (TGFB) protein array. We found that TBBPA increased cell proliferation and promoted fibrosis in 3D ht-UtLM spheroids with increased deposition of collagens. TBBPA upregulated the expression of profibrotic genes and corresponding proteins associated with the TGFB pathway. TBBPA activated TGFB signaling through phosphorylation of TGFBR1 and downstream effectors-small mothers against decapentaplegic -2 and -3 proteins (SMAD2 and SMAD3). The 3D ht-UtLM spheroid model is an effective system for studying environmental agents on human uterine fibrosis. TBBPA can promote fibrosis in uterine fibroid through TGFB/SMAD signaling.

Comparison of 3D airway models for the assessment of fibrogenic chemicals.

Lung epithelial cells and fibroblasts play key roles in pulmonary fibrosis and are involved in fibrotic signaling and production of the extracellular matrix (ECM), respectively. Recently, 3D airway models consisting of both cell types have been developed to evaluate the fibrotic responses while facilitating cell-cell crosstalk. This study aimed to evaluate the fibrotic responses in these models using different fibrogenic agents, which are known as key events in adverse outcome pathways of pulmonary fibrosis. We quantified cell injury and several sequential steps in fibrogenesis, including inflammation, the epithelial-mesenchymal transition (EMT), fibroblast activation, and ECM accumulation, using two different 3D airway models, the EpiAirway™-full thickness (Epi/FT) and MucilAir™-human fibroblast (Mucil/HF) models. In the Epi/FT model, fibrogenic agents induced the expression of inflammation and EMT-associated markers, while in the Mucil/HF model, they induced fibroblast activation and ECM accumulation. Using this information, we conducted gene ontology term network analysis. In the Epi/FT model, the terms associated with cell migration and response to stimulus made up a large part of the network. In the Mucil/HF model, the terms associated with ECM organization and cell differentiation and proliferation constituted a great part of the network. Collectively, our data suggest that polyhexamethyleneguanidine phosphate and bleomycin induce different responses in the two 3D airway models. While Epi/FT was associated with inflammatory/EMT-associated responses, Mucil/HF was associated with fibroblast-associated responses. This study will provide an important basis for selecting proper 3D airway models and fibrogenic agents to further research or screen chemicals causing inhalation toxicity.

DOCK2 Promotes Pleural Fibrosis by Modulating Mesothelial to Mesenchymal Transition.

Mesothelial to mesenchymal transition (MesoMT) is one of the crucial mechanisms underlying pleural fibrosis, which results in restrictive lung disease. DOCK2 (dedicator of cytokinesis 2) plays important roles in immune functions; however, its role in pleural fibrosis, particularly MesoMT, remains unknown. We found that amounts of DOCK2 and the MesoMT marker α-SMA (α-smooth muscle actin) were significantly elevated and colocalized in the thickened pleura of patients with nonspecific pleuritis, suggesting the involvement of DOCK2 in the pathogenesis of MesoMT and pleural fibrosis. Likewise, data from three different pleural fibrosis models (TGF-ß [transforming growth factor-ß], carbon black/bleomycin, and streptococcal empyema) consistently demonstrated DOCK2 upregulation and its colocalization with α-SMA in the pleura. In addition, induced DOCK2 colocalized with the mesothelial marker calretinin, implicating DOCK2 in the regulation of MesoMT. Our in vivo data also showed that DOCK2-knockout mice were protected from Streptococcus pneumoniae-induced pleural fibrosis, impaired lung compliance, and collagen deposition. To determine the involvement of DOCK2 in MesoMT, we treated primary human pleural mesothelial cells with the potent MesoMT inducer TGF-ß. TGF-ß significantly induced DOCK2 expression in a time-dependent manner, together with α-SMA, collagen 1, and fibronectin. Furthermore, DOCK2 knockdown significantly attenuated TGF-ß-induced α-SMA, collagen 1, and fibronectin expression, suggesting the importance of DOCK2 in TGF-ß-induced MesoMT. DOCK2 knockdown also inhibited TGF-ß-induced Snail upregulation, which may account for its role in regulating MesoMT. Taken together, the current study provides evidence that DOCK2 contributes to the pathogenesis of pleural fibrosis by mediating MesoMT and deposition of neomatrix and may represent a novel target for its prevention or treatment.

Aloin alleviates pathological cardiac hypertrophy via modulation of the oxidative and fibrotic response.

AIMS: Pathological cardiac hypertrophy is a characteristic feature in many cardiovascular diseases (CVDs). Aloin is an anthraquinone glycoside from Aloe species, and the effect of aloin on cardiac hypertrophy and associated fibrotic changes have not been elucidated. This study investigated the effect of aloin against the isoproterenol (ISO)-induced cardiac hypertrophy in rats. MAIN METHODS: Cardiac hypertrophy experimental model was induced in rats by subcutaneous injection of ISO for 14 days. Meanwhile, the animals were administered orally with aloin at doses of 25 and 50 mg/kg/day. On the 15th day, cardiac echocardiography was performed, the heart was collected and subjected for histopathological, gene expression, and immunoblot studies. Additionally, the effect of aloin on ISO-induced hypertrophic changes in H9c2 cells was investigated. KEY FINDINGS: Aloin markedly alleviated ISO-induced heart injury, reduced cardiac hypertrophy, improved cardiac function, and histological alterations in the heart. Mechanistically, aloin attenuated ISO-induced fibrosis via inhibition of the levels of collagen I, α-smooth muscle actin (α-SMA), fibronectin, transforming growth factor-ß (TGF-ß) and pSmad2/3 proteins in the heart. Aloin alleviated ISO-induced myocardial oxidative damage and up-regulated the levels of antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins. Moreover, aloin treatment attenuated ISO-induced hypertrophic changes and the generation of reactive oxygen species (ROS) in H9c2 cells in vitro. SIGNIFICANCE: Our findings demonstrated that aloin alleviated ISO-induced cardiac hypertrophy and fibrosis via inhibiting TGF-ß/pSmad2/3 signaling and restoring myocardial antioxidants, and therefore has promising therapeutic potential against cardiac hypertrophy and fibrosis.

The mTOR inhibitor everolimus attenuates tacrolimus-induced renal interstitial fibrosis in rats.

AIMS: Tacrolimus-a widely used immunosuppressant to prevent allograft rejection after organ transplantation-is nephrotoxic, increasing the risk of kidney injury accompanied by kidney fibrosis. The mammalian target of rapamycin (mTOR) inhibitor, everolimus, is an immunosuppressant used together with tacrolimus. Although mTOR signaling inhibition has been demonstrated to exhibit antifibrotic effects, the efficacy of everolimus against tacrolimus-induced kidney fibrosis has not been explored. Therefore, we evaluated the protective effects of everolimus against tacrolimus-induced kidney fibrosis. MAIN METHODS: To assess antifibrotic effect of everolimus against tacrolimus-induced kidney fibrosis, male Wistar rats were subcutaneously administered vehicle or tacrolimus (5 mg/kg per day) and/or everolimus (0.2 mg/kg per day) for 2 weeks after bilateral renal ischemia for 45 min. The antifibrotic effect of everolimus was also assessed using rat kidney fibroblast cell line (NRK-49F). KEY FINDINGS: Tacrolimus administration increased predominant profibrotic cytokine transforming growth factor-ß (TGF-ß) and fibroblast activation marker α-smooth muscle actin (α-SMA) expression and promoted the infiltration of macrophages in the kidney cortex, resulting in renal interstitial fibrosis in rats. Tacrolimus increased serum creatinine, blood urea nitrogen, kidney injury molecule-1 (KIM-1), and kidney injuries, such as tubular dilation, vacuolization, and glomerular atrophy. Everolimus administration attenuated tacrolimus-induced kidney fibrosis and the associated abnormalities. Everolimus strongly suppressed TGF-ß-induced kidney fibroblast activation and extracellular matrix protein expression by the mTOR signaling inhibition. SIGNIFICANCE: We demonstrated that everolimus attenuates tacrolimus-induced renal interstitial fibrosis in rats. Owing to its protective effect against tacrolimus-induced kidney fibrosis, everolimus may be useful when used concomitantly with tacrolimus.

Design, synthesis and activity evaluation of prodrug form JBP485 and Vitamin E for alleviation of NASH.

Non-alcoholic steatohepatitis (NASH) is a serious form of non-alcoholic fatty liver disease (NAFLD) characterized by liver steatosis with lobular inflammation, hepatocyte injury and pericellular fibrosis. JBP485 is a hydrophilic dipeptide with protective effects on liver through alleviation of oxidative stress and inhibition of hepatocyte apoptosis and ICAM-1 expression. Vitamin E (VE), as a powerful biological antioxidant, exerts a certain protective effect on cell membranes and lipoproteins from lipid peroxidation. In this study, on the basis of the structural characteristics of two agents, the prodrug form target of JBP485 and VE (JBP485-VE) was designed and synthesized via succinic acid linker. The synthesized compound significantly reduced the degree of inflammation and fibrosis according to hematoxylin-eosin (H&E) and sirius red staining assay for the liver tissue in CCl4-induced NASH mouse model. The clear reduction of TG, T-CHO and ALT, AST content also demonstrated its efficacy in the treatment of NASH. In addition, JBP485-VE also reduced the expression of the inflammatory markers IL-2, IL-17A and malondialdehyde (MDA) in liver tissue, which indicated its higher anti-inflammatory and anti-oxidative stress activity. All these evaluated biological properties suggest that the strategy of prodrug design provided an effective method for the treatment of NASH.

Indoxyl Sulfate Contributes to mTORC1-Induced Renal Fibrosis via The OAT/NADPH Oxidase/ROS Pathway.

Activation of mTORC1 (mechanistic target of rapamycin complex 1) in renal tissue has been reported in chronic kidney disease (CKD)-induced renal fibrosis. However, the molecular mechanisms responsible for activating mTORC1 in CKD pathology are not well understood. The purpose of this study was to identify the uremic toxin involved in mTORC1-induced renal fibrosis. Among the seven protein-bound uremic toxins, only indoxyl sulfate (IS) caused significant activation of mTORC1 in human kidney 2 cells (HK-2 cells). This IS-induced mTORC1 activation was inhibited in the presence of an organic anion transporter inhibitor, a NADPH oxidase inhibitor, and an antioxidant. IS also induced epithelial-mesenchymal transition of tubular epithelial cells (HK-2 cells), differentiation of fibroblasts into myofibroblasts (NRK-49F cells), and inflammatory response of macrophages (THP-1 cells), which are associated with renal fibrosis, and these effects were inhibited in the presence of rapamycin (mTORC1 inhibitor). In in vivo experiments, IS overload was found to activate mTORC1 in the mouse kidney. The administration of AST-120 or rapamycin targeted to IS or mTORC1 ameliorated renal fibrosis in Adenine-induced CKD mice. The findings reported herein indicate that IS activates mTORC1, which then contributes to renal fibrosis. Therapeutic interventions targeting IS and mTORC1 could be effective against renal fibrosis in CKD.