Omics approach to reveal the effects of obesity on the protein profiles of the exosomes derived from different adipose depots.

BACKGROUND: Obesity affects the cargo packaging of the adipocyte-derived exosomes. Furthermore, adipocytes in different adipose tissues have different genetic makeup, the cargo contents of the exosomes derived from different adipose tissues under obesity conditions should be different, and hence their impacts on the pathophysiological conditions. METHODS AND RESULTS: iTRAQ-based quantitative proteomics show that obesity has more prominent effects on the protein profiles of the exosomes derived from subcutaneous adipose tissue (SAT-Exos) in the high fat diet-induced obesity (DIO) mice than those derived from epididymal adipose tissue (EAT-Exos) and visceral adipose tissue (VAT-Exos). The differentially expressed proteins (DEPs) in SAT-Exos and VAT-Exos are mainly involved in metabolism. Subsequent untargeted metabolomic and lipidomics analyses reveal that injection of these SAT-Exos into the B6/J-Rab27a-Cas9-KO mice significantly affects the mouse metabolism such as fatty acid metabolism. Some of the DEPs in SAT-Exos are correlated with fatty acid metabolism including ADP-ribosylation factor and mitogen-activated protein kinase kinase kinase-3. Pathway analysis also shows that SAT-Exos affect adipocyte lipolysis and glycerophospholipid metabolism, which is in parallel with the enhanced plasma levels of fatty acids, diglycerides, monoglycerides and the changes in glycerophospholipid levels in DIO mice. CONCLUSION: Our data provide scientific evidence to suggest SAT-Exos contribute to the changes in plasma lipid profiles under obesity conditions.

Combination of artesunate and WNT974 induces KRAS protein degradation by upregulating E3 ligase ANACP2 and ß-TrCP in the ubiquitin-proteasome pathway.

BACKGROUND: KRAS mutation is one of the dominant gene mutations in colorectal cancer (CRC). Up to present, targeting KRAS for CRC treatment remains a clinical challenge. WNT974 (LGK974) is a porcupine inhibitor that interferes Wnt signaling pathway. Artesunate (ART) is a water-soluble semi-synthetic derivative of artemisinin. METHODS: The synergistic effect of ART and WNT974 combination in reducing CRC cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RT-PCR was utilized for the mRNA levels of KRAS, CUL7, ANAPC2, UBE2M, RNF123, SYVN1, or ß-TrCP. Western blot assay was utilized for the protein levels of NRAS, HRAS, KRAS, ANAPC2, ß-TrCP, GSK-3ß, p-Akt (Ser473), t-Akt, p-PI3K (Tyr458), t-PI3K, p-mTOR (Ser2448), t-mTOR. Xenograft mouse model assay was performed for the anti-CRC effect of combination of ART and WNT974 in vivo. IHC assay was utilized for the levels of KRAS, ß-TrCP, GSK-3ß or ANAPC2 in tumor tissues. RESULTS: Our study shows that the combination of WNT974 and ART exhibits synergistic effect in reducing CRC growth. The combination treatment significantly reduces KRAS protein level and activity in CRC cells. Interestingly, the combination treatment increases E3 ligases ANAPC2 expression. Our data show that overexpression of ANAPC2 significantly reduces KRAS protein levels, which is reversed by MG132. Knockdown of ANAPC2 in CRC abolishes the combination treatment-reduce KRAS expression. Besides, the treatment also increases the expressions of GSK-3ß and E3 ligase ß-TrCP that is known to degrade GSK-3ß-phosphorylated KRAS protein. Knockdown of ß-TrCP- and inhibition of GSK-3ß abolish the combination treatment-induce KRAS ubiquitination and reduction in expression. Last but not least, combination treatment suppresses PI3K/Akt/m-TOR signaling pathway. CONCLUSIONS: Our data clearly show that the combination treatment significantly enhances KRAS protein degradation via the ubiquitination ubiquitin-proteasome pathway, which is also demonstrated in xenograft mouse model. The study provides strong scientific evidence for the development of the combination of WNT974 and ART as KRAS-targeting therapeutics for CRC treatment. Video Abstract.

The impact of obesity on adipocyte-derived extracellular vesicles.

Recently, the emerging roles of adipocyte-derived extracellular vesicles (EVs) linking obesity and its comorbidities have been recognized. In obese subjects, adipocytes are having hypertrophic growth and are under stressed. The dysfunction adipocytes dysregulate the assembly of the biological components in the EVs including exosomes. This article critically reviews the current findings on the impact of obesity on the exosomal cargo contents that induce the pathophysiological changes. Besides, this review also summarizes the understanding on how obesity affects the biogenesis of adipocyte-derived exosomes and the exosome secretion. Furthermore, the differences of the exosomal contents in different adipose depots, and the impact of obesity on the exosomes that are derived from the stromal vascular fraction such as the adipose tissue macrophages and adipocyte-derived stem cells will also be discussed. The current development and potential application of exosome-based therapy will be summarized. This review provides crucial information for the design of novel exosome-based therapy for the treatment of obesity and its comorbidities.

Apigenin inhibits STAT3/CD36 signaling axis and reduces visceral obesity.

Visceral obesity is the excess deposition of visceral fat within the abdominal cavity that surrounds vital organs. Visceral obesity is directly associated with metabolic syndrome, breast cancer and endometrial cancer. In visceral obese subjects, signal transducer and activator of the transcription 3 (STAT3) in adipocytes is constitutively active. In this study, we aimed to screen for dietary herbal compounds that possess anti-visceral obesity effect. Apigenin is abundant in fruits and vegetables. Our data show that apigenin significantly reduces body weight and visceral adipose tissue (VAT), but not subcutaneous (SAT) and epididymal adipose tissues (EAT), of the high fat diet (HFD)-induced obese mice. Mechanistic studies show that HFD increases STAT3 phosphorylation in VAT, but not in SAT and EAT. Further studies suggest that apigenin binds to non-phosphorylated STAT3, reduces STAT3 phosphorylation and transcriptional activity in VAT, and consequently reduces the expression of STAT3 target gene cluster of differentiation 36 (CD36). The reduced CD36 expression in adipocytes reduces the expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) which is the critical nuclear factor in adipogenesis. Our data show that apigenin reduces CD36 and PPAR-γ expressions and inhibits adipocyte differentiation; overexpression of constitutive active STAT3 reverses the apigenin-inhibited adipogenesis. Taken together, our data suggest that apigenin inhibits adipogenesis via the STAT3/CD36 axis. Our study has delineated the mechanism of action underlying the anti-visceral obesity effect of apigenin, and provide scientific evidence to support the development of apigenin as anti-visceral obesity therapeutic agent.

Palmitic acid is an intracellular signaling molecule involved in disease development.

Emerging evidence shows that palmitic acid (PA), a common fatty acid in the human diet, serves as a signaling molecule regulating the progression and development of many diseases at the molecular level. In this review, we focus on its regulatory roles in the development of five pathological conditions, namely, metabolic syndrome, cardiovascular diseases, cancer, neurodegenerative diseases, and inflammation. We summarize the clinical and epidemiological studies; and also the mechanistic studies which have identified the molecular targets for PA in these pathological conditions. Activation or inactivation of these molecular targets by PA controls disease development. Therefore, identifying the specific targets and signaling pathways that are regulated by PA can give us a better understanding of how these diseases develop for the design of effective targeted therapeutics.

Simultaneous determination of wogonin, oroxylin a, schisandrin, paeoniflorin and emodin in rat serum by HPLC-MS/MS and application to pharmacokinetic studies.

Wogonin and oroxylin A in Scutellariae Radix, schisandrin in Chinensis Fructus, paeoniflorin in Moutan Cortex and emodin in Polygoni Cuspidate Rhizome et Radix are anti-inflammatory active compounds. A method for simultaneous determination of the five compounds in rat was developed and validated using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The separation was performed on a Symmetry C18 column (4.6 × 50 mm, 3.5 µm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The detection was performed using multiple-reaction monitoring with electrospray ionization source in positive-negative ion mode. The calibration curves showed good linearity (r ≥ 0.9955). The lower limit of quantification (LLOQ) was 5 ng/mL for wogonin and schisandrin, 10 ng/mL for oroxylin A and emodin, and 15 ng/mL for paeoniflorin, respectively. The relative standard deviations of intraday and interday precisions were <11.49 and 14.28%, respectively. The extraction recoveries and matrix effects were acceptable. The analytes were stable under the experiment conditions. The validated method has been successfully applied to pharmacokinetic studies of the five compounds in rats after oral administration of Hu-gan-kan-kang-yuan capsule. This paper would be a valuable reference for pharmacokinetic studies of Chinese medicine preparations containing the five compounds.

Developing an Absorption-Based Quality Control Method for Hu-Gan-Kang-Yuan Capsules by UFLC-QTOF-MS/MS Screening and HPLC-DAD Quantitative Determination.

Traditional Chinese Medicine Preparations (TCMPs) contain massive numbers of ingredients responsible for their multiple efficacies. An absorption-based quality control method for complicated TCMPs using Hu-gan-kang-yuan Capsule (HGKYC) as an example was developed. To select proper chemical markers for quality control of HGKYC, an ultra-fast liquid chromatography (UFLC) coupled with electrospray ionization quadrupole time-off light mass spectrometry (UFLC-QTOF-MS/MS) method was used for the rapid separation and structural identification of the constituents in the HGKYC extract and the rat serum after oral administration of HGKYC. As a result, one hundred and seven prototype constituents including flavonoids, organic acid, phenylpropanoids, anthraquinones, saponins, alkaloids, terpenes, phenols and amino acids in HGKYC extract, and 43 compounds found in rat serum after oral administration of HGKYC were unambiguously identified or tentatively characterized by comparing retention times and MS information with those of authentic standards or available literature references. Finally, a simple, low-cost and effective method of simultaneous determination for baicalein, wogonin, paeonol and emodin in HGKYC was developed using high performance liquid chromatography coupled with a diode array detector. In conclusion, an absorption-based quality control pattern was developed and successfully used for evaluating HGKYC.

Ankylosing spondylitis and glaucoma in European population: A Mendelian randomization study.

Background: The relationship between ankylosing spondylitis (AS) and glaucoma in the European population remains unclear. In the present study, we applied a two-sample Mendelian randomization (MR) method to investigate their causal relationship. Methods: MR analysis was conducted to validate the causal associations between AS with glaucoma using summary statistics from the genome-wide association studies of AS (9,069 cases and 13,578 control subjects) and glaucoma (8,591 cases and 210,201 control subjects). The inverse variance weighting method was performed to evaluate the causal relationship. The MR-Egger regression approach was applied to assess pleiotropy, while Cochran's Q test was used to analyze heterogeneity. Subgroup analysis was performed according to primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG). Results: The results of the MR study reveal a risk-increasing causal relationship between AS and glaucoma among European populations (OR = 1.35, 95%CI = 1.16-1.57, P = 8.81 × 10-5). Pleiotropy and heterogeneity were not found in our study. In the subgroup analysis, AS was also causal with POAG (OR = 1.48, 95%CI = 1.17-1.86, P = 8.80 × 10-4) and PACG (OR = 1.91, 95%CI = 1.03-3.51, P = 3.88 × 10-2). Conclusion: The results of the MR analysis suggested a causal relationship between AS and glaucoma in the European population. Further studies are needed to identify the specific mechanism between these two diseases.

Roles and Mechanisms of Regulated Necrosis in Corneal Diseases: Progress and Perspectives.

Regulated necrosis is defined as cell death characterized by loss of the cell membrane integrity and release of the cytoplasmic content. It contributes to the development and progression of some diseases, including ischemic stroke injury, liver diseases, hypertension, and cancer. Various forms of regulated necrosis, particularly pyroptosis, necroptosis, and ferroptosis, have been implicated in the pathogenesis of corneal disease. Regulated necrosis of corneal cells enhances inflammatory reactions in the adjacent corneal tissues, leading to recurrence and aggravation of corneal disease. In this review, we summarize the molecular mechanisms of pyroptosis, necroptosis, and ferroptosis in corneal diseases and discuss the roles of regulated necrosis in inflammation regulation, tissue repair, and corneal disease outcomes.

Surgical Management and Prognosis of Congenital Choledochal Cysts in Adults: A Single Asian Center Cohort of 69 Cases.

BACKGROUND: The choledochal cyst (CC) is a rare cystic dilatory condition with malignant tendency, which is more frequently reported in children. Surgical resection of cysts can significantly decrease the risk of malignancy and reduce associated complications. However, CC has been paid lesser attention in adults, and its surgical parameters have been frequently reported to be in dispute. This study aimed to report experience associated with the treatment of an adult with CC and to suggest the appropriate parameters for the surgery, including the extent of excision (complete or not), the length of the Y limb, the diameter of the cholangio-intestinal anastomosis (CIA), and different operative approaches (open, laparoscopic, and laparoscopic converted to open) by comparing the various indicators, including postoperative bile leakage, cholangitis, choledocholithiasis, carcinogenesis, and surgical re-excision. METHODS: We conducted a single-center noninterventional retrospective study of 69 different congenital choledochal cyst patients who were admitted to our hospital between July 2010 and July 2020. We collected and analyzed their demographic data, clinical presentations, underlying complications, imaging tests, endoscopic interventions, and parameters for the surgery, histological data, and prognostic indicators over a mean 77-month follow-up period. RESULTS: We found that out of the 69 cases, the median age at diagnosis was 32 (IQR = 22-45) years. Seven (10.1%) patients were asymptomatic before the diagnosis, with abdominal pain as the primary complaint in 62 (89.9%) patients, whereas nausea/vomiting was observed in 29 (42.0%) patients. CCs were mainly evaluated by using magnetic resonance cholangiopancreatography (MRCP) (n = 47, 68.1%). It was observed that surgery, cholecystectomy, choledochal cysts excision, and Roux-en-Y hepaticojejunostomy (n = 65, 94.2%), and laparotomy (n = 58, 84.1%) were the dominant therapeutic modalities employed. However, seventeen (24.6%) patients were treated with incomplete cyst resection, while 52 (75.4%) patients received complete cyst resection. We also conducted regular follow-ups after the surgery for a mean duration of 77 months. Postoperative complications were found to be experienced by 35 (50.7%) patients, and a further two patients (2.9%) developed malignancy during the follow-up. Moreover, increasing the diameter of cholangio-intestinal anastomosis served as a potential protective factor for postoperative choledocholithiasis (p = 0.040) and a risk factor for cholangitis (p = 0.002). CONCLUSIONS: Among the 69 CC participants, abdominal pain was their major symptom. Those with an abnormal pancreaticobiliary junction were more likely to have choledocholithiasis and pancreatitis. The diagnosis was found to be highly dependent on the Todani classification scheme and MRCP. Surgical resection remains the key to CC treatment. The results suggested that the complete resection, the length of the Y limb of 40 cm-60 cm, and the diameter of the CIA of 1.0 cm-1.5 cm were appropriate values for predicting the lower risk of postoperative complications.

Discovery of a novel anti-obesity meroterpenoid agent targeted subcutaneous adipose tissue.

BACKGROUND: Meroterpenoid furanasperterpene A (T2-3) with a novel 6/6/6/6/5 pentacyclic skeleton was isolated from the Aspergillus terreus GZU-31-1. Previously, we showed that T2-3 possessed significant lipid-lowering effects in 3T3-L1 adipocytes at 5 µM concentration. However, its therapeutic effect in metabolic disease and the underlying mechanisms of action remain unclear. METHODS: High fat diet-induced obesity (DIO) mouse model and 3T3-L1 cell model were used to assess the anti-obesity effects of T2-3. Lipids in the adipocytes were examined by Oil Red O staining. ß-catenin expression was examined by immunofluorescence and Western blotting, its activity was assessed by TOPflash/FOPflash assay. RESULTS: T2-3 possessed potent anti-obesity effects in DIO mice, it significantly reduced body weight and subcutaneous adipose tissue (SAT) mass. Mechanistic studies showed that T2-3 significantly inhibited 3T3-L1 preadipocyte differentiation as indicated by the reduced number of mature adipocytes. The treatments also reduced the expressions of critical adipogenic transcription factors CEBP-α and PPAR-γ in both 3T3-L1 adipocytes and SAT in DIO mice. Interestingly, T2-3 increased the cytoplasmic and nuclear expressions of ß-catenin and the transcriptional activity of ß-catenin in 3T3-L1 adipocytes; the elevated ß-catenin expression was also observed in SAT of the T2-3-treated DIO mice. Indeed, upregulation of ß-catenin activity suppressed adipogenesis, while ß-catenin inhibitor JW67 reversed the anti-adipogenic effect of T2-3. Taken together, our data suggest that T2-3 inhibits adipogenesis by upregulating ß-catenin activity. CONCLUSIONS: Our study is the first report demonstrating meroterpenoid furanasperterpene A as a novel 6/6/6/6/5 pentacyclic skeleton (T2-3) that possesses potent anti-adipogenic effect by targeting ß-catenin signaling pathway. Our findings drive new anti-obesity drug discovery and provide drug leads for chemists and pharmacologists.

Diagnosis and Prognosis of Retroperitoneal Liposarcoma: A Single Asian Center Cohort of 57 Cases.

BACKGROUND: Liposarcoma is a soft tissue malignancy, commonly observed in the extremities. However, retroperitoneal liposarcoma is seldom reported and its diagnosis is frequently neglected. This study aims to present the clinical characteristics, diagnosis, and prognosis of five liposarcoma subtypes and report our experience of patient treatment. METHODS: We conducted a single-center noninterventional retrospective study of 57 retroperitoneal liposarcoma patients admitted to Peking Union Medical College Hospital (PUMCH, Beijing, China) between July 2011 and December 2019. We collected and analyzed their demographic, clinical, imaging, histological, therapeutic, and prognostic data over a mean 4.5-year follow-up period. RESULTS: Twenty-five (44%) patients were asymptomatic prior to diagnosis, with abdominal distension as the chief complaint in 18 (32%) patients and abdominal pain observed in 16 (28%) patients. Masses were evaluated by computed tomography (n = 48, 84%) or ultrasound (n = 25, 44%). Laparotomy (n = 52, 91%) was the dominant therapeutic modality rather than laparoscopy (n = 5, 9%). All patients were treated with R0 resection except two patients who underwent R2 resection. We conducted regular follow-ups every six months after surgery for a mean duration of 4.5 years. Recurrence was experienced by 14 (25%) patients and a further 9 (16%) died during follow-up. CONCLUSIONS: Abdominal distension and pain are chief complaints with liposarcoma. As the extremities are the main liposarcomas locations, the diagnosis of retroperitoneal liposarcoma is usually neglected. Since half of the patients are asymptomatic, timely diagnosis and treatment are highly dependent on regular ultrasound and computed tomography imaging. R0 resection is the key to retroperitoneal liposarcoma treatment. In comparison, patients who underwent R2 resection, which is considered a palliative treatment, had bad prognoses. Large, symptomatic dedifferentiated, and pleomorphic liposarcomas are more likely to have poor prognoses, while the prognosis for well-differentiated or myxoid liposarcoma is relatively good.

Dihydroartemisinin is potential therapeutics for treating late-stage CRC by targeting the elevated c-Myc level.

Currently, no frontline treatment is effective for the late-stage colorectal cancer (CRC). Understanding the molecular differences in different stages of CRC can help us to identify the critical therapeutic targets for designing therapeutic strategy. Our data show that c-Myc protein is highly expressed in late-stage CRC when compared with early-stage CRC in both clinical samples and in cell lines representing different cancer stages. Given that c-Myc is a well-known oncogenic driver in CRC, its high expression in the late-stage CRC may represent a critical therapeutic target for treating the cancer. Dihydroartemisinin treatment significantly increases c-Myc protein degradation and hence reduces its expression in CRC. The treatment also reduces CRC cell viability. Interestingly, dihydroartemisinin exhibits a more potent growth-inhibitory effect in late-stage CRC than the early-stage CRC. The treatment also possesses potent growth-inhibitory effects in mouse models bearing c-Myc-overexpressed CRC. The reduced c-Myc level and its reduced transcriptional activity reduce the expressions of acetyl-CoA carboxylase, fatty acid synthase, carnitine-palmitoyltransferase-1, and medium-chain acyl-CoA dehydrogenase in the cancer cells. Lipidomics study also shows that dihydroartemisinin treatment changes the metabolic phenotypes in CRC, reduces oxygen consumption, respiration, and ATP production, hence reduces the cell proliferation and induces apoptosis. Our study provides strong pharmacological evidence to support the translation of dihydroartemisinin for the treatment of late-stage CRC by targeting c-Myc.

Toll-like receptor 4 is a master regulator for colorectal cancer growth under high-fat diet by programming cancer metabolism.

Although high-fat diet (HFD) has been implicated in the development of colorectal cancer (CRC), the critical signaling molecule that mediates the cancer growth is not well-defined. Identifying the master regulator that controls CRC growth under HFD can facilitate the development of effective therapeutics for the cancer treatment. In this study, the global lipidomics and RNA sequencing data show that, in the tumor tissues of CRC-bearing mouse models, HFD not only increases tumor weight, but also the palmitic acid level and TLR4 expression, which are reduced when HFD is replaced by control diet. These concomitant changes suggest the roles of palmitic acid and TLR4 in CRC growth. Subsequent studies show that palmitic acid regulates TLR4 expression in PU.1-dependent manner. Knockdown of PU.1 or mutations of PU.1-binding site on TLR4 promoter abolish the palmitic acid-increased TLR4 expression. The role of palmitic acid/PU.1/TLR4 axis in CRC growth is further examined in cell model and animal models that are fed either HFD or palmitic acid-rich diet. More importantly, iTRAQ proteomics data show that knockdown of TLR4 changes the metabolic enzyme profiles in the tumor tissues, which completely abolish the HFD-enhanced ATP production and cancer growth. Our data clearly demonstrate that TLR4 is a master regulator for CRC growth under HFD by programming cancer metabolism.

Combination of Wogonin and Artesunate Exhibits Synergistic anti-Hepatocellular Carcinoma Effect by Increasing DNA-Damage-Inducible Alpha, Tumor Necrosis Factor α and Tumor Necrosis Factor Receptor-Associated Factor 3-mediated Apoptosis.

Hepatocellular carcinoma (HCC) is difficult to treat, and is the second leading cause of cancer-related death worldwide. This study aimed to examine whether combination of wogonin and artesunate exhibits synergistic anti-HCC effect. Our data show that the combination treatment exhibits synergistic effect in reducing HCC cell viability by increasing apoptosis as indicated by the elevated cleavage of caspase 8, 3 and PARP. Interestingly, PCR array and the subsequent studies indicate that the combination treatment significantly increases the expression of DNA-damage-inducible, alpha (GADD45A), tumor necrosis factor (TNFα) and TNF receptor-associated factor 3 (TRAF3). Knockdown of GADD45A, TNFα or TRAF3 abolishes the combination treatment-enhanced apoptosis and the synergistic effect in reducing HCC cell viability. In the HCC-bearing xenograft mouse models, although the combination treatment increases the activity of NFκB in the tumor tissues, it exhibits a more potent anti-HCC effect than the mono-treatment, which may due to the enhanced apoptosis as indicated by the increased expression of GADD45A, TNFα, TRAF3 and apoptotic markers. Our study clearly demonstrates that the combination of artesunate and wogonin exhibits synergistic anti-HCC effect, and support the further development of this combination as alternative therapeutics for HCC management.

The Therapeutic Effects and Possible Mechanism of Pranoprofen in Mouse Model of Corneal Alkali Burns.

OBJECTIVE: To investigate therapeutic effects and possible mechanism of pranoprofen in a mouse model of corneal alkali burns and provide new evidence for the clinical treatment of corneal alkali burns. METHODS: A unilateral alkali burn was created in the central cornea by placing a piece of 2 mm diameter filter paper soaked in 1N NaOH on the right eye for 30 seconds. After the model was performed, C57BL/6J mice received topical treatment with saline eye drops or pranoprofen eye drops and were, respectively, categorized as saline group and pranoprofen group, whereas the remaining normal mice that were not subjected to alkali burns served as control, each group containing 15 mice (n = 45). On the 5th day after model establishment, the corneal fluorescein sodium staining score was evaluated in order to assess corneal epithelial damage. Tissue HE stain was used to observe the pathological changes of corneal tissue in each group. Real-time RT-PCR and western blot were also performed to detect the mRNA and protein expression of NLRP3, IL-1ß/p17, and matrix metallopeptidase MMP-13. RESULTS: 5 days after burns, microscopic observations of the pranoprofen group showed less corneal opacity and neovascularization development than the saline group. Sodium fluorescein staining showed obvious corneal structure disorders, poor corneal epithelium continuity, and a larger corneal epithelial defect area in the saline group (10.33±+-0.57) as opposed to the pranoprofen group (8.33 ± 0.57） (p < 0.05). HE stain results showed the saline group had obvious corneal structure disorder and the corneal epithelial layer was incomplete as opposed to the pranoprofen group. PCR and western blot results suggested that the pranoprofen group expressed less NLRP3, IL-1ß, and MMP-13 mRNA and protein expression in corneal tissue than the saline group (p < 0.05). CONCLUSION: Pranoprofen may alleviate inflammatory response by inhibiting the expression levels of NLRP3 and IL-1ß at the early stage of corneal alkali injury, lowering the expression of MMP-13 and ultimately reducing corneal epithelial damage.

Endogenous Toll-like Receptor 2 Modulates Th1/Treg-Promoting Dendritic Cells in Mice Corneal Transplantation Model.

PURPOSE: Endogenous toll-like receptor (TLR) 2 is linked to allograft rejection in corneal transplantation. TLR2 also could modulate dendritic cell (DC) phenotype, resulting in T cell polarization. Thus, we investigated the role of endogenous TLR2 on DC development and T cell polarization during corneal rejection. MATERIALS AND METHODS: Corneas of BALB/c mice were transplanted into the eyes of C57BL/6 wild-type (WT) recipients and TLR2-/- (KO) recipients. Graft survival and TLR2 mRNA expression were assessed. At day 14 after transplantation, to study endogenous TLR2 effects on DC development and function, surface expression of MHC classⅡ (MHCⅡ), CD86, CD80 and CD40 in ipsilateral cervical draining lymph nodes (DLNs) is measured by flow cytometry, and DC phenotype in corneas is detected by immunofluorescence. The levels of IL-12, IL-10 and IL-4 in corneas were measured by real time-qPCR (RT-qPCR). The ability of DCs to stimulate T cell polarization was assessed by IFN-γ expressions via RT-qPCR and immunohistochemistry. RESULTS: TLR2 mRNA expression in corneas was peaked at day 14 post-transplantation in WT group. KO group improved corneal allograft survival compared to the WT group. In addition, the KO group decreased expression of CD86, CD80 and CD40 on DCs compared to the WT group. There was no difference in MHCⅡ expression in two groups. The CD11c+MHCⅡ+CD40high DCs could not be detected in corneas of the KO group. Moreover, the KO group decreased IL-12 (Th1-promoting cytokines) mRNA expression and increasing IL-10 (Treg-promoting cytokines) mRNA expression compared to the WT group. IL-4 (Th2-promoting cytokines) mRNA expression gained no difference between the two groups. The IFN-γ (Th1 cytokines) expression was significantly decreased in the KO group compared to the WT group. CONCLUSIONS: Endogenous TLR2 may contribute to allogeneic corneal rejection via Th1 immunity by activating Th1-promoting DCs and suppressing Treg-promoting DCs.

LncRNAs in ocular neovascularizations.

The prevalence of eye diseases worldwide is dramatically increasing and represents a major concern in underdeveloped and developed regions. Ocular diseases, previously associated with a higher depression risk, also impose a substantial economic burden on affected families, thus early detection and/or accurate treatment in order to avoid and prevent blindness should be emphasized. Ocular neovascularization (NV), the leading cause of blindness in a variety of eye diseases, is a pathologic process characterized by the formation, proliferation and infiltration of anomalous, tiny and leaky fragile blood vessels within the eye. Genetics have been suspected to play an important role in the occurrence of eye diseases, with the detection of a numbers of specific gene mutations. Long non-coding RNA (lncRNAs) are novel class of regulatory molecules previously associated with various biological processes and diseases, however the nature of the relation and pathways by which they might contribute to the development of corneal, choroidal and retinal NV have not yet been completely elucidated. In this review, we focus on the regulation and characteristics of lncRNAs, summarize results from ocular NV-related studies and discuss the implication of lncRNAs in ocular NV development.

Effects of moderate soil salinity on osmotic adjustment and energy strategy in soybean under drought stress.

Under drought and soil salinity, plants usually respond to accumulate inorganic and organic osmolytes for adaptation, that would induce changes in energy consumption strategy of plants. Moderate soil salinity would enable plants to lower energy consumption for osmotic adjustment by passively absorbing more Na+. This action would keep more energies for growth of drought-stressed plants. Thus, Na+ accumulation might be an energy-efficient strategy for plants to cope with drought was speculated. To support this speculation, we assessed the effects of soil salinity on osmotic adjustment and energy utilization under drought in this study. Our results indicated that the ratio and content of inorganic osmolytes was significantly higher under drought-saline stress (D + S) than those under single drought stress (D), while the osmolarity and contents of organic osmolytes of D + S were significantly lower than those of D. This indicated that moderate soil salinity could enable soybean seedlings to consume relatively lower energies to produce less organic osmolytes and accumulate more inorganic ions for osmotic adjustment coping with drought. Meanwhile the water content, cell turgor, ash content, and specific leaf area and biomass of D + S were significantly higher than those of D, but the leaf construction cost of D + S was significantly lower than those of D. This suggested that moderate soil salinity could enhance water retention, and reduce the photoassimilate and energy consumption of droughty soybean seedlings. This work would help to understand the positive effects of moderate soil salinity on plant growth on the level of osmotic adjustment and energy consumption strategy.