TGF-β1 mRNA Expression Research Articles

Effects of Lactobacillus plantarum HFY15 on Lupus Nephritis in Mice by Regulation of the TGF-β1 Signaling Pathway.

ObjectiveIn this study, the Lactobacillus plantarum HFY15 (LP-HFY15) strain isolated from naturally fermented yak yogurt was investigated. An animal model of lupus nephritis was established by pristane to verify the interventional effect of LP-HFY15 on mouse lupus nephritis by regulating the transforming growth factor-β1 (TGF-β1) signaling pathway.Materials and MethodsIndexes in mouse serum and tissues were detected by kits, pathological changes in mouse kidney were observed by hematoxylin-eosin (H&E) staining, and quantitative polymerase chain reaction (qPCR) was used to detect TGF-β 1-related expression in mouse kidney tissue, which further elucidated the mechanism of LP-HFY15.ResultsLP-HFY15 decreased the elevation of urinary protein and the levels of interleukin-6 (IL-6), IL-12, tumor necrosis factor alpha (TNF-α), and interferon γ (IFN-γ) in serum and kidney tissue. LP-HFY15 also reduced serum creatinine (SCr), blood urea nitrogen (BUN), total cholesterol (TC), triglyceride (TG), and raised total protein (TP), and albumin (ALB) levels in mice with nephritis. In addition, LP-HFY15 inhibited the positive rate of double-stranded deoxyribonucleic acid (dsDNA) antibodies in mice with nephritis. The observation of H&E sections showed that LP-HFY15 alleviated the glomerulus morphological incompleteness and inflammatory infiltration caused by nephritis. Further results showed that LP-HFY15 downregulated the mRNA expression of TGF-β1, vascular endothelial growth factor (VEGF), and nuclear factor kappa-B (NF-κB) in the kidneys of lupus nephritis mice, and the expression of inhibitor of NF-κB (IκB-α), copper/zinc superoxide dismutase (Cu/Zn-SOD), and manganese superoxide dismutase (Mn-SOD) was also upregulated.ConclusionThese results indicated that LP-HFY15 plays a significant role in experimental intervention for lupus nephritis. The effect of LP-HFY15 was positively correlated with its concentration, and the effect was similar to that of prednisone at 109 CFU/kg.

Effects of dietary fermented Chinese herbal medicines on growth performance, digestive enzyme activity, liver antioxidant capacity, and intestinal inflammatory gene expression of juvenile largemouth bass (Micropterus salmoides)

This study aimed to evaluate the effects of fermented Chinese herbal medicines (FCHM) on growth performance, digestive enzyme activity, antioxidant capacity and intestinal inflammatory gene expression of juvenile largemouth bass (Micropterus salmoides). Four trial diets H0 (basal diet), H1, H2, and H3 (added 1 %, 3 %, and 5 % FCHM to the basal diet, respectively) were prepared. Fish were randomly divided into 4 treatment groups of 3 replicates per treatment (20 fish/replicate) and reared for 8 weeks. The results of the test showed that: 1) Feeding 1 % FCHM significantly increased the weight gain rate (P < 0.01) and specific growth rate (P < 0.05) of juvenile largemouth bass compared with the control group. 2) Compared with the control group, FCHM had no significant effect on the body composition of juvenile largemouth bass (P > 0.05) but could result in an overall decreasing trend in muscle drip loss, steaming loss, and freezing loss. 3) FCHM significantly increased the activity of SOD, GSH-PX and CAT in the liver of juvenile largemouth bass and decreased the MDA compared to that of the control group (P < 0.01). (4) Compared with the control group, FCHM significantly increased the relative mRNA expression of the intestinal anti-inflammatory factors TGF-β and IL-10 (P < 0.01) and decreased the pro-inflammatory factors IL-8, IL-15 (P < 0.01) and TNF-α (P < 0.05) of juvenile Largemouth bass. In summary, a 1 % FCHM addition amount can improve the growth performance, liver antioxidant and immune capacity of juvenile largemouth bass.

Vasohibin-2 promotes proliferation and metastasis of cervical cancer cells by regulating epithelial-mesenchymal transition

To explore the role of vasohibin-2 (VASH2) in regulation of proliferation and metastasis of cervical cancer cells. We analyzed the differentially expressed genes between cervical cancer cells with flotillin-1 overexpression and knockdown by RNA-seq combined with analysis of public databases. The expression levels of VASH2 were examined in normal cervical epithelial cells (HcerEpic), cervical cancer cell lines (HeLa, C-33A, Ca ski, SiHa and MS751) and fresh cervical cancer tissues with different lymph node metastasis status. We further tested the effects of lentivirus-mediated overexpression and interference of VASH2 on proliferation, migration, invasion and lymphatic vessel formation of the cervical cancer cells and detected the expression levels of key epithelial-mesenchymal transition (EMT) markers and TGF-β mRNA. RNA-seq and analysis of public databases showed that VASH2 expression was significantly upregulated in cervical cancer cells exogenously overexpressing flotillin-1 (P < 0.05) and downregulated in cells with flotillin-1 knockdown (P < 0.05), and was significantly higher in cervical cancer tissues with lymph node metastasis than in those without lymph node metastasis (P < 0.01). In cervical cancer cell lines Ca Ski, SiHa, and MS751 and cervical cancer tissue specimens with lymph node metastasis, VASH2 expression was also significantly upregulated as compared with HcerEpic cells and cervical cancer tissues without lymph node metastasis (P < 0.05). Exogenous overexpression of VASH2 significantly promoted proliferation, migration, invasion and lymphatic vessel formation of cervical cancer cells, whereas these abilities were significantly inhibited in cells with VASH2 knockdown (P < 0.05). The cervical cancer cells overexpressing VASH2 showed significant down- regulation of e-cadherin and up- regulation of N-cadherin, Vimentin and VEGF-C, while the reverse changes were detected in cells with VASH2 knockdown (P < 0.05). TGF-β mRNA expression was significantly up-regulated in cervical cancer cells overexpressing VASH2 and down-regulated in cells with VASH2 knockdown (P < 0.001). Flotillin-1 may participate in TGF-β signaling pathway-mediated EMT through its down-stream target gene VASH2 to promote the proliferation, migration, invasion and lymphatic vessel formation of cervical cancer cells in vitro.

Exercise Augments the Effect of SGLT2 Inhibitor Dapagliflozin on Experimentally Induced Diabetic Cardiomyopathy, Possible Underlying Mechanisms.

One of the most prevalent cardiovascular problems linked with type 2 diabetes mellitus (T2DM) is diabetic cardiomyopathy (DCM). DCM is associated with myocardial oxidative stress, inflammation, apoptosis, suppressed autophagy, extracellular matrix remodeling, and fibrosis. The current study aims to investigate the protective effect of sodium-glucose transport 2 inhibitor (SGLT2i) dapagliflozin and/or exercise on DCM. Thirty adult male Sprague Dawley rats are used. T2DM is induced by a 6-week high-fat diet (HFD) followed by a single intraperitoneal (IP) injection of 35 mg/kg streptozotocin (STZ). Rats are divided into five groups, control, diabetic (DM), DM + swimming, DM + dapagliflozin, and DM + dapagliflozin and swimming. Serum glucose, insulin, insulin resistance (HOMA-IR), and cardiac enzymes (CK-MB and lactate dehydrogenase (LDH) are measured. Heart specimens are used for evaluation of cellular oxidative stress markers malondialdehyde (MDA), antioxidant enzymes, glutathione (GSH), and catalase (CAT), as well as mRNA expression of TGF-β, MMP9, IL-1β, and TNF-α. Stained sections with haematoxylin and eosin (H & E) and Masson trichrome are used for histopathological evaluation and detection of fibrosis, respectively. Immunohistochemical staining for apoptosis (caspase-3), and autophagy (LC3) are also carried out. The combinations of SGLT2i and exercise exhibited the most significant cardioprotective effect. It improved diabetic-induced histopathological alterations in the myocardium and attenuated the elevation of serum blood glucose, CK-MB, LDH, myocardial MDA, and mRNA expression of TNF-α, IL-1β, TGF-β, MMP9, and the immune expression of caspase-3. Moreover, this combination increased the serum insulin, myocardial antioxidants GSH and CAT, and increase the immune expression of the LC-3. In conclusion, a combination of SGLT2i and exercise exerted a better antioxidant, anti-inflammatory, and antifibrotic effect in DCM. Moreover, the combination enhances the autophagic capacity of the heart.

Synovitis Ointment Improved Knee Osteoarthritis by Suppressing SDF-1/CXCR4 Signaling Pathway.

Objective Knee osteoarthritis (KOA) remains a challenge for clinicians worldwide and lacks major advancements in treatment. In this study, we investigated the mechanism of synovitis ointment interference on KOA. Methods SD rats were used to establish KOA models and were randomly divided into five groups: the control group, the KOA group, the KOA + synovitis ointment group, the KOA + Western medicine group, and the KOA + Chinese medicine group. Detection of pathological injury of the joint was observed through HE staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression of SDF-1, CXCR4, MMP-9, and MMP-13. Effects of synovitis ointment on bone cell fibrosis were detected through Masson staining, and the relative mRNA expression of PLOD2, COL1A1, TIMP1, and TGF-β was observed using the real-time quantitative (RT-PCR) method. Results Mankin's score and the knee diameters showed that the KOA model has been successfully established; compared with the OA group, the synovitis ointment group improved the pathological injury of the knee joint. Compared with the KOA group, the synovitis ointment group, the KOA + Western medicine group, and the KOA + Chinese medicine group significantly decreased the expression of SDF-1, CXCR4, MMP-9, and MMP-13. Synovitis ointment reduced the relative content of bone cell fiber compared to that in the KOA group. While, the relative mRNA expression of PLOD2, COL1A1, TIMP1, and TGF-β was significantly decreased in the synovitis ointment group. Conclusion Synovitis ointment inhibited the inflammation and bone cell fibrosis of KOA, and the mechanism was related to the SDF-1/CXCR4 singling pathway.

Ganoderma lucidum polysaccharide ameliorated diabetes mellitus-induced erectile dysfunction in rats by regulating fibrosis and the NOS/ERK/JNK pathway

BackgroundDiabetes mellitus-induced erectile dysfunction (DMED) is a frequent complication of diabetes mellitus (DM), with limited therapy at present. This study aimed to explore the role and mechanism of Ganoderma lucidum polysaccharide (GLP) on DMED.MethodsDMED was induced in the experimental rats [male 12-week-old Sprague-Dawley (SD) rats] by treatment with streptozotocin (60 mg/kg) and apomorphine (APO). Next, rats in the GLP low dose (GLP-L)/GLP high dose (GLP-H) groups were treated with GLP (100 or 400 mg/kg/d, respectively) for 8 weeks. Subsequently, erectile function was assessed by APO and electrostimulation of the cavernous nerve (CN). Serum or penile testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and cyclic guanosine monophosphate (cGMP) contents were evaluated by enzyme-linked immunosorbent assay (ELISA). The levels of oxidative stress indicators in the corpus cavernosum (CC) were measured by corresponding kits, and histological changes in the CC were observed by hematoxylin-eosin (HE) and Masson staining. Additionally, the apoptosis index, caspase-3, caspase-9, and eNOS expression, and mitochondrial membrane potential (MMP) were also detected. Furthermore, quantitative polymerase chain reaction (qPCR) and western blot assays were conducted to determine the NOS, TGF-β1 mRNA expression, ERK1/2, eNOS, JNK phosphorylation, and arginase II protein expression.ResultsThe erectile function test revealed that erectile dysfunction (ED) was alleviated in the DMED rats following treatment with GLP. Moreover, GLP upregulated the T and cGMP content, improved the oxidative stress and histological injuries of CC, and also inhibited the apoptosis and MMP loss of penile tissues in DMED rats. Furthermore, GLP treatment enhanced the mRNA expression of NOS and TGF-β1 and suppressed the phosphorylation of ERK1/2, eNOS, and JNK, as well as the protein expression of arginase II in DMED rats.ConclusionsGLP ameliorated DMED by repairing the CC pathological damage and upregulating NOS expression and ERK/JNK phosphorylation, indicating that GLP may be a candidate drug for DMED therapy.

Abstract LB022: A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues

Abstract While most cases of cutaneous squamous cell carcinoma (cSCC) are benign, invasive cSCC is associated with higher mortality and is often more difficult to treat. As such, understanding the factors that influence the progression of cSCC are important. Aggressive cancers metastasize through a series of evolutionary changes, collectively called the epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells transition to a highly mobile mesenchymal cell type with metastatic capacities. While changes in expression of TGF-β, vimentin, and E-cadherin are hallmarks, EMT is not an “all or none” process. Using patient-derived cSCC and adjacent normal tissues (ANT), we observed a significant increase in mRNA expression of TGF-β (p&amp;lt;0.05), TGF-βR2 (p&amp;lt;0.001), and E-cadherin (p&amp;lt;0.005) in cSCC tissues by qPCR. Immunoblots performed on homogenized tissues yielded significant and parallel increases in protein expression of TGF-β (p&amp;lt;0.05) and E-cadherin (p&amp;lt;0.05). We also observed increased expression of the EMT-associated transcription factors Zeb1 and Snai1 in cSCC tissue sections using immunohistochemistry. Interestingly, we observed a significant increase in expression of vimentin (p&amp;lt;0.01) by immunoblot and differential expression of four oncogenic and EMT-associated miRNAs; miR-31 expression was significantly higher in cSCC tissues (p&amp;lt;0.001), while we observed a significant decrease in expression of miR-34a (p&amp;lt;0.05), miR-200b, and miR-200c in cSCC tissues. Examination of ANT and cSCC tissue sections using immunofluorescence and immunohistochemistry showed differential, and reverse, expression of E-cadherin and vimentin among cells within an individual tumor. Collectively, our data support a hypothesis that cSCC tumors are undergoing a hybrid EMT program, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. This hybrid EMT program may be promoting both survival and invasiveness of the tumors. A better understanding of this hybrid EMT process may influence therapeutic strategies in more invasive disease. Citation Format: Christopher S. Pulford, Chandana K. Uppalapati, McKale R. Montgomery, Elizabeth E. Hull, Kathryn J. Leyva. A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB022.

Tanshinone IIA regulates the TGF-β1/Smad signaling pathway to ameliorate non-alcoholic steatohepatitis-related fibrosis.

Tanshinone IIA (TIIA) is a major component extracted from the traditional herbal medicine Salvia miltiorrhiza and has been indicated to play a role in the treatment of organ fibrosis. However, the evidence supporting its antifibrotic effect is insufficient and the underlying mechanism is unclear. To investigate the therapeutic effect of TIIA on non-alcoholic steatohepatitis-related fibrosis (NASH-F), the present study used a methionine choline deficiency diet to induce NASH-F in rats, and explored the effect of TIIA on the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway. Wistar rats were randomly divided into control, NASH-F and TIIA groups. After 8 weeks of treatment, the levels of serum markers associated with liver function and fibrosis were measured, liver fat vacuoles and inflammation were assessed by haematoxylin and eosin staining, and liver fibrosis was assessed by Masson's trichrome staining. TGF-β1, Smad2, Smad3, Smad7 and α-smooth muscle actin (α-SMA) mRNA expression, and TGF-β1, Smad2/3, phosphorylated (p)-Smad2/3, Smad7 and α-SMA protein levels were determined. The results revealed that TIIA could remarkably ameliorate liver fat vacuoles and inflammation in NASH-F rats, and could decrease the levels of serum aspartate aminotransferase, alanine aminotransferase, total bilirubin, total bile acid, hyaluronic acid, type Ⅳ collagen, laminin and type III collagen, while increasing the levels of total cholesterol and triglycerides; however, this was not statistically significance. TIIA markedly suppressed the increased TGF-β1, Smad2, Smad3 and α-SMA mRNA expression levels observed in the liver of NASH-F rats, while it increased the mRNA expression level of Smad7. Similarly, TIIA suppressed the increased TGF-β1, p-Smad2/3 and α-SMA protein levels observed in the liver of NASH-F rats, while it increased the protein expression level of Smad7 in vitro and in vivo. TIIA had no significant cytotoxic effect at 10, 20, 40 and 80 µmol/l on human LX-2 cell. In conclusion, the findings of the present study indicated that TIIA alleviated NASH-F by regulating the TGF-β1/Smad signaling pathway. TIIA may be a useful tool in the prevention and treatment of NASH-F.

Concomitant Expression of IL-6 and TGF-β Cytokines and their Receptors in Peripheral Blood of Patients with Multiple Sclerosis: The Effects of INFβ Drugs.

Concomitant signals from IL-6 and TGF-β have a central role in the Th17 cells development and differentiation, and these cells are the main promoters of demyelinating inflammation in the central nervous system (CNS) resulting in multiple sclerosis (MS). To evaluate the simultaneous IL-6 and TGF-β gene and their receptor protein expression in patients with Relapsing-Remitting (RR)-MS. IL-6 and TGF-β mRNA and their receptor expression on the surface of CD4+T cells were evaluated using real-time PCR (RT-PCR) and flow cytometry, respectively. The IL-6 mRNA expression in patients with RRMS was significantly higher than in the controls (p= 0.019). When patients who did not receive any other treatment were compared with the controls, the significant difference was substantial (p=0.006). The TGF-β mRNA expression in patients was lower than in the controls (p = 0.03). However, in patients receiving IFNβ, it increased compared with the other patients (p= 0.036). There was no difference in cytokine receptor expression between patients and the control group. Our data conclude an increase and decrease in mRNA expression levels of IL-6 and TGF-β in patients with RRMS, respectively. Moreover, there were no significant differences in receptor expression of either cytokines. Based on our data the balance of TGF and IL-6 appears to have a positive impact on the disease control.

The protective effect of collagen peptides from bigeye tuna (Thunnus obesus) skin and bone to attenuate UVB-induced photoaging via MAPK and TGF-β signaling pathways

The aim of this study was to investigate the protective effect and mechanism of bigeye tuna (Thunnus obesus) skin collagen peptides (TSCP) and bone collagen peptides (TBCP) on skin photoaging. In UVB-damaged NIH/3T3 cells, TSCP and TBCP could effectively scavenge excessive reactive oxygen species, suppress mRNA and protein expression of MMP-1, and inhibit the decreased mRNA and protein expression of TGF-β1 and type I collagen. In the ICR mice model exposed to UVB, TSCP and TBCP could effectively improve moisture content, Hyp content and total antioxidant capacity level, activate TGF-β1 signaling pathway, and inhibit MAPK signaling pathway, thereby retarding UVB-induced skin photoaging. TSCP exhibited more excellent antioxidant activity, inhibited p-p38 activation and promoted TGF-β1 expression via TGF-β signaling pathway. By contrast, TBCP was more potent to down-regulate protein expression of p-ERK, p-JNK and MMP-1. The present work provides a theoretical basis for employment of collagen peptides to attenuate UVB-induced photoaging.

Correlation of TNF-α, IFN-γ and TGF-β Expression with In-vitro Fertilization Success Rates in Women with Recurrent Implantation Failure Undergoing Treatment with Intra-lipid Infusion

This study aimed at assessing the correlations between gene expression of TNF-α, IFN-γ, TGF-β in peripheral lymphocytes from women suffering from repeated implantation failure before and after intravenous Intra-lipid (IL) therapy, and correlation between changes in gene expression with IL infusion and success rate of IVF cycles. Twenty-three women complaining of unexplained infertility without history of autoimmune disorders, or immunodeficient diseases were included. All women included aged &lt;40 years, BMI &lt;28 with history of recurrent IVF cycles failure, seeking medical advice for new IVF trial. All were average responders to induction of ovulation (≥5 oocytes in each cycle) with good quality embryos transferred to uterus at proper time. Included women received 200 ml of 10% IL slowly intravenous. Two venous blood samples were taken from all candidates, one before IL infusion and the second was at day of embryo transfer. The current study detected a significant reduction of expression in TNF-α and increased expression of TGF-β, while non-significant reduction in expression of IFN-γ after treatment. Significant associations between reduction of TNF-α, IFN-γ expression and positive clinical and ongoing pregnancy were observed, while increased TGF-β expression was associated with only positive ongoing pregnancy. In conclusion, IL therapy might have a positive impact on IVF pregnancy rates via alterations in peripheral cytokines expression mainly reduction of TNF-α mRNA expression and increased TGF-β mRNA expression.

Effect of Bushen Qudu Decoction on TGF-β1/Smads signal transduction pathway in rats with chronic renal failure

Purpose: To study the impact of Bushen Qudu Decoction on renal function, renal tissue morphology and TGF-β1/Smads signal transduction pathway in adenine-induced chronic renal failure rats.Methods: 76 rats were assigned to normal group, model group, uremic clearance group, benazepil group, Bushen Qudu decoction gunshan A group and Bushen Qudu decoction Guiza group. Renal failure was induced in rats using intragastric administration of adenine for 30 days. The expressions of TGF-β1, Smad2, Smad3 and Smad6 were determined with immunohistochemistry. Real-time quantitative PCR was employed to assay the mRNA expression of TGF-β1 in each group, while protein expressions of Smad2, Smad3 and Smad6 were determined with western blot. The scores of glomerular and tubulointerstitial lesions in each group were obtained by histopathological examination.Results: Bushen Qudu decoction significantly reduced BUN and creatinine in rats with chronic renal failure; furthermore, it also lowered the protein expressions of TGF-β1 and SMAD2/3, but Smad6 protein, relative to control (p &lt; 0.05). The TGF-β1mRNA expression was down-regulated (p &lt; 0.05), relative to control group.Conclusion: Bushen Qudu decoction reduces renal interstitial fibrosis in rats and delays the progression of chronic renal failure by regulating TGF-β1/Smads signaling pathway. These findings provide some insight that should facilitate the development of new drugs that would delay the onset of chronic renal failure.

Amelioration of aluminum-induced hepatic and nephrotoxicity by Premna odorata extract is mediated by lowering MMP9 and TGF-β gene alterations in Wistar rat

This study aims to investigate the effect of Premna odorata (P. odorata) (Lamiaceae) on the hepatic and nephrotoxicity induced by aluminum chloride (AlCl3) in rat. Wistar male rats were equally classified into four groups: control, P. odorata extract (500 mg/kg B.W.), AlCl3 (70 mg/kg B.W.), and P. odorata extract plus AlCl3 groups. All treatments were given orally for 4 weeks. Serum transaminases and some biochemical parameters, hepatic and renal antioxidant/oxidant biomarker; tumor necrosis factor-α (TNF-α); matrix metalloproteinase (MMP9) and transforming growth factor-β (TGF-β) mRNA expression; histopathological examination of the liver, and kidneys were investigated. The obtained results revealed that AlCl3 significantly increased the activities of serum aspartate transaminase, alanine transaminase, and alkaline phosphatase as well as produced a significant increase in total cholesterol, triglyceride, urea, and creatinine concentrations, while there were no changes observed in the total protein, albumin, and globulin concentrations. Also, aluminum administration significantly decreased the reduced glutathione content and increased the catalase activity, malondialdehyde, and TNF-α concentrations in the liver and kidney tissue. Moreover, AlCl3 results in congestion, degeneration, and inflammation of the liver and kidney tissue. Co-treatment of P. odorata extract with AlCl3 alleviated its harmful effects on the previous parameters and reduced the histopathological alterations induced by AlCl3. Therefore, Premna odorata may have a potent protective effect against oxidative stress induced by Al toxicity through downregulation of MMP9 and TGF-β gene expression.

Evaluation of IGF-1, TNF-α, and TGF-β Gene Expression after Oral Vitamin D Supplementation in School-Aged Children with Chronic Bronchial Asthma

BACKGROUND: Airway remodeling in children with bronchial asthma is due to the effect of inflammatory mediators and growth factors on the bronchial epithelium. Vitamin D (VitD) has immunomodulatory effect in many inflammatory diseases as bronchial asthma. The ant-inflammatory and anti-fibrotic role of VitD could prevent or improve air way remodeling in asthmatic patients. AIM: The study investigated the effect of VitD supplementation on the expression of transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and insulin growth factor 1(IGF-1) and to correlate them with asthma severity and level of control. METHODS: The serum level of VitD and the mRNA expression of IGF-1, TGF-β, and TNF-α were estimated in 50 patients and 20 healthy controls control subjects using quantitative PCR in real-time. Asthmatic patients with VitD deficiency received VitD supplementation for 2 months followed by remeasurement of serum VitD and the genes expression TGF-β, TNF-α, and IGF-1. RESULT: Pre-intake of VitD and serum level of VitD were lower in all patients than control subjects (p = 0.005). VitD level was directly correlated with IGF-1 mRNA expression, which was indirectly correlated with TGF-β, r = 0.5 and −0.57; p = 0.0001 and 0.002, respectively. After VitD supplementation, the expression of the TGF-β mRNA gene was the only gene that decreased significantly (p = 0.04) together with improved asthma control and spirometric parameters. CONCLUSIONS: VitD supplementation down regulated the gene expression of TGF-β and improved asthma control level, but it did not significantly affect the gene expression of TNF-α and IGF-1.

Sex specific effects of buprenorphine on behavior, astrocytic opioid receptor expression and neuroinflammation after pediatric traumatic brain injury in mice

Children who suffered traumatic brain injury (TBI) often experience acute and chronic pain, which is linked to a poor quality of life. Buprenorphine (BPN) is commonly used to treat moderate to severe persistent pain in children, however, the efficacy and safety profile of BPN in the pediatric population is still inconclusive. This study investigated the sex-specific effects of BPN on body weight, motor coordination and strength, expression of opioid receptors in the white matter astrocytes, and neuroinflammation in a mouse impact acceleration model of pediatric TBI. Male and female littermates were randomized on postnatal day 20–21(P20-21) into Sham, TBI + saline and TBI + BPN groups. Mice in the TBI + saline and TBI + BPN groups underwent TBI, while the Sham group underwent anesthesia without injury. BPN (0.075 mg/kg) was administered to the TBI + BPN mice at 30 min after injury, and then every 6–12 h for 2 days. Mice in the TBI + saline group received the same amount of saline injections. The impact of BPN on body weight, motor function, opioid receptor expression, and neuroinflammation was evaluated at 1-day (d), 3-d and 7-d post-injury. We found that 1) TBI induced significant weight loss in both males and females. BPN treatment improved weight loss at 3-d post-injury in females. 2) TBI significantly impaired motor coordination and strength. BPN improved motor coordination and strength in both males and females at 1-d and 3-d post-injury. 3) TBI significantly decreased exploration activity at 1-d post-injury in males, and at 7-d post-injury in females, while BPN improved the exploration activity in females. 4) TBI significantly increased mRNA expression of mu-opioid receptors (MOR) at 7-d post-injury in males, but decreased mRNA expression of MOR at 1-d post-injury in females. BPN normalized MOR mRNA expression at 1-d post-injury in females. 5) MOR expression in astrocytes at corpus callosum significantly increased at 7-d post-injury in male TBI group, but significantly decreased at 1-d post-injury in female TBI group. BPN normalized MOR expression in both males and females. 6) TBI significantly increased the mRNA expression of TNF-α, IL-1β, IL-6 and iNOS. BPN decreased mRNA expression of iNOS, and increased mRNA expression of TGF-β1. In conclusion, this study elucidates the sex specific effects of BPN during the acute phase after pediatric TBI, which provides the rationale to assess potential effects of BPN on chronic pathological progressions after pediatric TBI in both males and females.

Sanguinarine attenuates hydrogen peroxide-induced toxicity in liver of Monopterus albus: Role of oxidative stress, inflammation and apoptosis

In aquatic animals, hydrogen peroxide (H2O2), which is a source of oxidative stress, can cause physiological dysfunction, inflammation, and death. Sanguinarine (SAN) is a plant extract known to improve antioxidant and immune capacity. However, the roles of SAN in H2O2-induced liver tissue toxicity is unclear. The effects on hepatic oxidative damage, inflammatory response, and apoptosis were investigated by feeding rice field eel with 0, 375, and 750 μg/kg of SAN for eight weeks and then intraperitoneally injected with H2O2. The results showed that after 24 h of H2O2 injection, the activities of ALT and AST in serum were significantly increased, oxidative damage and inflammatory response occurred in the liver, and apoptosis was induced, which indicated that H2O2 induced liver damage in rice field eel. However, dietary supplemented with 375 or 750 μg/kg SAN significantly decreased the activities of ALT and AST in serum, and significantly increased the antioxidant function (decreased ROS, MDA, and antioxidant enzymes levels, downregulated antioxidant-related gene expression, and inhibited the transcription level of nrf2). The addition of SAN at 375 or 750 μg/kg ameliorated H2O2-induced inflammatory response of liver (upregulated tgf-β1 mRNA expression, downregulated il-1β, il-6, il-8, and il-12β mRNA expression, and inhibited the transcription levels of tlr-3 tlr-7, and nf-κb). In addition, dietary supplemented with 375 or 750 μg/kg SAN alleviated the apoptosis of liver induced by H2O2 (downregulated bax mRNA expression, upregulated caspase3 mRNA expression, and reduced the number of apoptotic cells by TUNEL staining). Overall, these results suggested that SAN could alleviate the liver injury in rice field eel induced by H2O2, mainly by improving antioxidant capacity, alleviating inflammatory response and inhibiting apoptosis, and the effect of 750 μg/kg SAN addition is better than 375 μg/kg.

MO441: Dynamic Role of Macrophage-Inducible C-Type Lectin in the Process of AKI-to-CKD Transition

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is a global health concern associated with high morbidity and mortality, as well as a high risk of progression to chronic kidney disease (CKD). As a result of phenotype diversity and plasticity, macrophages are recognized as key regulators of kidney repair, regeneration, and fibrosis. Mincle (macrophage-inducible C-type lectin, Clec4e) was reported to promote and maintain inflammatory response in AKI. However, the specific role of Mincle in the AKI-to-CKD transition is still unclear. METHOD In this study, WT and Mincle-/-mice were subjected to renal ischemia-reperfusion (I/R) injury (ischemia for 35 min) without resecting the right kidney and were analyzed on days 1, 3, 14 and 30 to explore the potential effects and mechanism of Mincle on renal inflammation and fibrosis. RNA-seq was performed on day 30 to detect underlying pathogenesis. RESULTS Our results showed that after I/R injury, renal interstitial macrophages identified by F4/80 immunofluorescence staining were up-regulated from day 1. Then macrophages gradually increased over time and obviously infiltrated in injured kidney both on day 14 and day 30. Mincle was colocalized to renal macrophages in WT mice but was not detectable in Mincle-/- mice. RT-PCR suggested that Mincle start to increase on day 1 and reached the highest level on day 14, and then decreased on day 30 which was consistent with the immunofluorescence of Mincle. During the I/R induced AKI-to-CKD progression, we observed similar dynamic change patterns for proinflammatory cytokines and fibrosis markers and Mincle expression in WT mice. Of note, on day 30, we found that Mincle deletion aggravated renal inflammatory response as detected by inflammatory cytokines like MCP-1, IL-6 and IL-1β. Renal fibrosis also deteriorated in Mincle-/- mice with increased mRNA expression of TGF-β, fibronectin and collagen I. Quantitative Masson staining and immunofluorescent α-SMA further confirmed more kidney fibrosis in Mincle-/-mice than WT mice. Then we performed gene set enrichment analysis (GSEA) of the differentially expressed genes in two groups on day 30 and identified enriched toll-like receptor signaling and NF-κ-B signaling in Mincle-/-mice indicating more activated inflammatory responses. RNA-seq also showed top 5 significantly up-regulated genes (Slpi, Cd79a, Jchain, Lyz1 and Mzb1) related to the immune response in the context of Mincle deficiency, among which secretory leukocyte protease inhibitor (SLPI) showed 60-fold higher expression compared with WT mice. Next, increased SLPI mRNA expression was confirmed by RT-PCR in I/R kidney in Mincle-/-mice compared to the WT group. And increased SLPI positive cells in fibrotic kidneys after I/R at day 14 and day 30 in Mincle-/-mice was also observed, suggesting its potential role in renal fibrosis caused by Mincle deletion. CONCLUSION To conclude, Mincle may exert a sustaining effect on the long-term outcome of the AKI-to-CKD transition through modulating inflammation and fibrosis. Clarifying the specific function of Mincle positive macrophages during the process of AKI-to-CKD transition may provide new insight into the underlying mechanism of CKD progression.

Association of the Glucocorticoid Receptor Gene Polymorphisms with TGF-β1 mRNA Expression in Childhood Asthma

Background: Glucocorticoid (GC) is a fundamental drug used to treat asthma. GC binds to its corresponding receptor (GR) to formulate a complex that increases the production of anti-inflammatory factors and decreases the amount of pro-inflammatory mediators, covering many cytokines. GR is a nuclear receptor superfamily protein, encoded by NR3C1 gene. Studies suggest that polymorphisms of the NR3C1 gene contribute to a decreased response to GC for the treatment of asthma, even leading to drug-resistance. Also, TGF-β1 plays a central role in airway remodeling, GC significantly inhibits the production of TGF-β1, and TGF-β1 can induce GC resistance. Thus, it is possible that the polymorphisms of the NR3C1 gene can affect the expression of TGF-β1 mRNA and tissue remodeling. Objectives: This study evaluates the effect of polymorphisms (TthIII1, BclI, ER22/23EK, and N363S) of the NR3C1 GR gene on TGF-β1 mRNA expression in children with asthma. Methods: The samples of this study included 52 outpatients (age range: 6 - 14 years) with asthma referred to Huai’an First People’s Hospital, Nanjing Medical University, from January 2018 to June 2019. Meanwhile, 40 healthy volunteers were included as the control group. Results: The polymorphisms of the NR3C1 GR gene were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and TGF-β1 mRNA levels were measured by real-time reverse transcription (RT)-PCR. TthIII1 and TGF-β1 mRNA expression levels had significant (P = 0.011) correlations. But BclI showed no e effect on TGF-β 1 mRNA, N363S, and ER22/23EK had not been examined. Conclusions: According to the results, there was a relationship between single nucleotide polymorphisms (SNPs) of the NR3C1 gene and TGF-β1 mRNA in asthmatic children. TthIII1 CC and CT genotype have the strongest induction effect on the expression of TGF-1. The phenomenon suggests that SNPs may be involved in the asthma pathology.

Effect of androgen replacement therapy on erectile function through histological recovery in castrated rats

ABSTRACT Introduction Androgens are essential for physical activity in males and normal erectile function. Reportedly, androgen deficiency causes erectile dysfunction through tissue damage in the corpus cavernosum. We previously reported that delayed androgen replacement therapy improved erectile function in rats through histological recovery. Objective To investigate the mechanism of the recovery process by androgen replacement therapy in rats. Methods Twelve-week-old male Wistar ST rats were used in this study. The rats were assigned to the following groups: castrated (Cast), castrated with subcutaneous administration of testosterone undecanoate (25 mg/kg; Cast+T), and sham (Sham). Cast+T rats received subcutaneous doses of testosterone, beginning from 4 weeks after castration, whereas Sham and Cast rats received only the vehicle. Erectile function was assessed by evaluating intracavernosal pressure (ICP) and mean arterial pressure (MAP) after electrical stimulation of the cavernous nerve at 1, 2, 4, and 8 weeks after testosterone administration. mRNA expression of histological factors (Cavin-1, Cavin-2, Cavin-3, α-SMA, VEGF-R, ICAM-1, and TGF-β1) was tested using real-time PCR. Results The ratio of ICP to MAP was 0.26 ± 0.04 in the 4th week for the Cast group, and the erectile function was significantly lower than that of the Sham group (0.61 ± 0.03; p &amp;lt;0.05). On the other hand, in the Cast+T group, it changed over time from 0.41 ± 0.06 in the 1st week, 0.44 ± 0.04 in the 2nd week, 0.48 ± 0.04 in the 4th week, to 0.70 ± 0.03 in the 8th week, and erectile function was significantly improved 8 weeks after the testosterone administration (p &amp;lt;0.05). The Cavin-1 and Cavin-2 mRNA expressions were increased in the Cast+T group compared to the Cast group at all times (P &amp;lt;0.01). α-SMA, VEGF-R, ICAM-1, and TGF-β1 mRNA expressions were also increased in the Cast+T group, as compared to the Cast group (P &amp;lt;0.01). Conclusions In this study, we identified that androgen replacement therapy in rats would improve ED. This may be because the administration of testosterone improved histological recovery. It has been reported that these caveolae-related factors control the invagination structure of the cell membrane, and that caveolae in endothelial cells are also involved in the production of nitric oxide (NO), which might contribute to the therapeutic effect of androgen replacement therapy. Increase in the expression of these caveolae-related factors may regenerate smooth muscle and corpus cavernosum structures. Disclosure Work supported by industry: no.

Immunophenotyping of Acute Inflammatory Exacerbations of Lung Injury Driven by Mutant Surfactant Protein-C: A Role for Inflammatory Eosinophils.

Acute inflammatory exacerbations (AIEs) represent immune-driven deteriorations of many chronic lung conditions, including COPD, asthma, and pulmonary fibrosis (PF). The first line of therapy is represented by broad-spectrum immunomodulation. Among the several inflammatory populations mobilizing during AIEs, eosinophils have been identified as promising indicators of an active inflammatory exacerbation. To better study the eosinophil-parenchymal crosstalk during AIE-PF, this work leverages a clinically relevant model of inflammatory exacerbations triggered by inducible expression of a mutation in the alveolar epithelial type 2 cell Surfactant Protein-C gene [SP-CI73T]. Unbiased single-cell sequencing analysis of controls and SP-CI73T mutants at a time coordinated with peak eosinophilia (14 days) defined heightened inflammatory activation, chemotaxis, and survival signaling (IL-6, IL-4/13, STAT3, Glucocorticoid Receptor, mTOR, and MYC) in eosinophils. To study the impact of eosinophils in inflammatory exacerbations, the SP-CI73T line was crossed with eosinophil lineage deficient mice (GATA1Δdbl) to produce the SP-CI73TGATA1KO line. Time course analysis (7–42 days) demonstrated improved lung histology, survival, and reduced inflammation in SP-CI73TGATA1KO cohorts. Spectral flow cytometry of tissue digests confirmed eosinophil depletion in GATA1KO mice and the absence of a compensatory shift in neutrophils and immature monocyte recruitment. Eosinophil deletion resulted in progressive monocyte-derived macrophage accumulation (14 days post-injury), combined with declines in CD3+CD4+ lymphocyte and B220+ B cell abundance. Histochemical analysis revealed atypical inflammatory cell activation in SP-CI73TGATA1KO mice, with reduced numbers of Arg-1+ and iNOS+ cells, but increases in tgfb1 mRNA expression in bronchoalveolar lavage cells and tissue. Dexamethasone treatment (1 mg/kg daily, i.p.) was utilized to investigate corticosteroid efficacy in highly eosinophilic exacerbations induced by mutant SP-CI73T. Dexamethasone successfully reduced total and eosinophil (CD11b+SigF+CD11c−) counts at 14 days and was linked to reduced evidence of structural damage and perivascular infiltrate. Together, these results illustrate the deleterious role of eosinophils in inflammatory events preceding lung fibrosis and demonstrate the efficacy of corticosteroid treatment in highly eosinophilic exacerbations induced by mutant SP-CI73T.