Tag-free glycosylated rhMFG-E8 as a therapy for acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) can result from renal ischemia and reperfusion (I/R) and often occurs during surgical procedures in cardiac, liver, kidney transplantation and trauma-hemorrhage. Milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. Since MFG-E8 promotes clearance of apoptotic cells, we have explored its therapeutic potential in various organ injury conditions. To develop human MFG-E8 as a potential therapy, we have generated a human cell-expressed, and thus glycosylated, tag-free recombinant human (rh) MFG-E8 and tested its safety and biological activity in vitro. We hypothesize that the tag-free glycosylated rhMFG-E8 is protective in I/R-induced AKI and it can be developed as an effective therapy for AKI. METHODS: To assess the pharmacokinetic properties of the tag-free rhMFG-E8, Sprague Dawley rats were either untreated or treated with a bolus dose of the tag-free rhMFG-E8, blood collected at various time points and the recovery of human MFG-E8 in the blood were measured by ELISA. Adult male C57BL6 mice underwent bilateral renal ischemia for 30 min and immediately upon reperfusion, mice were treated intraperitoneally with either normal saline (vehicle) or 20 µg/kg human cell expressed, glycosylated tag-free rhMFG-E8. At either 24 h or 48 h after I/R, blood and kidneys were harvested for further analysis. In separate cohorts of mice after I/R and treatment, mice were observed for 10 days, and survival recorded. RESULTS: AKI rats treated with the tag-free rhMFG-E8 had similar half-life as those in the treated control rats. At 48 h after I/R-induced AKI, renal function markers, BUN and creatinine were increased and treatment with the tag-free rhMFG-E8 significantly decreased these markers. At both 24 h and 48 h after AKI, inflammatory cytokines, TNF-α, IL-6 and IL-1ß were increased and treatment decreased these levels. The kidney mRNA expressions of these cytokines were also increased at 24 h after AKI and treatment significantly decreased those mRNA expressions. Histologically, at 48 h after AKI, tubular damage, and the number of TUNEL staining cells were increased and treatment markedly decreased these measurements. Administration of tag-free rhMFG-E8 at the time of reperfusion improved survival in a 10-day survival study. CONCLUSION: Our new human cell-expressed tag-free rhMFG-E8 is protective in I/R-induced AKI and it may have the potential to be further developed as a safe and effective therapy for AKI.

FePd Nanozyme- and SKN-Encapsulated Functional Lipid Nanoparticles for Cancer Nanotherapy via ROS-Boosting Necroptosis.

Cell necroptosis has presented great potential, acting as an effective approach against tumor apoptotic resistance, and it could be further enhanced via accompanying reactive oxygen species (ROS) overexpression. However, whether overproduced ROS assists the necroptotic pathway remains unclear. Thus, iron-palladium nanozyme (FePd NZ)- and shikonin (SKN)-encapsulated functional lipid nanoparticles (FPS-LNPs) were designed to investigate the ROS overexpression-enhanced SKN-induced necroptosis. In this system, SKN acts as an effective necroptosis inducer for cancer cells, and FePd NZ, a sensitive Fenton reaction catalyst, produces extra-intracellular ROS to reinforce the necroptotic pathway. Both in vitro and in vivo antitumor evaluation revealed that FPS-LNPs presented the best tumor growth inhibition efficacy compared with FP-LNPs or SKN-LNPs alone. Meanwhile, induced necroptosis by FPS-LNPs can further trigger the release of damage-associated molecular patterns (DAMPs) and antigens from dying tumor cells to activate the innate immune response. Taking biosafety into consideration, this study has provided a potential nanoplatform for cancer nanotherapy via inducing necroptosis to avoid apoptosis resistance and activate CD8+ T cell immune response.

In situ neutrophil apoptosis and macrophage efferocytosis mediated by Glycyrrhiza protein nanoparticles for acute inflammation therapy.

In the progression of acute inflammation, the activation and recruitment of macrophages and neutrophils are mutually reinforcing, leading to amplified inflammatory response and severe tissue damage. Therefore, to regulate the axis of neutrophils and macrophages is essential to avoid tissue damage induced from acute inflammatory. Apoptotic neutrophils can regulate the anti-inflammatory activity of macrophages through the efferocytosis. The strategy of in situ targeting and inducing neutrophil apoptosis has the potential to modulate macrophage activity and transfer anti-inflammatory drugs. Herein, a natural glycyrrhiza protein nanoparticle loaded with dexamethasone (Dex@GNPs) was constructed, which could simultaneously regulate neutrophil and macrophage function during acute inflammation treatment by combining in situ neutrophil apoptosis and macrophage efferocytosis. Dex@GNPs can be rapidly and selectively internalized by neutrophils and subsequently induce neutrophils apoptosis through a ROS-dependent mechanism. The efferocytosis of apoptotic neutrophils not only promoted the polarization of macrophages into anti-inflammatory state, but also facilitated the transfer of Dex@GNPs to macrophages. This enabled dexamethasone to further modulate macrophage function. In mouse models of acute respiratory distress syndrome and sepsis, Dex@GNPs significantly ameliorated the disordered immune microenvironment and alleviated tissue injury. This study presents a novel strategy for drug delivery and inflammation regulation to effectively treat acute inflammatory diseases.

A NOVEL OLIGONUCLEOTIDE MRNA MIMIC ATTENUATES HEMORRHAGE-INDUCED ACUTE LUNG INJURY.

ABSTRACT: Hemorrhagic shock (HS) is accompanied by a pronounced activation of the inflammatory response in which acute lung injury (ALI) is one of the most frequent consequences. Among the pivotal orchestrators of this inflammatory cascade, extracellular cold-inducible RNA-binding protein (eCIRP) emerges as a noteworthy focal point, rendering it as a promising target for the management of inflammation and tissue injury. Recently, we have reported that oligonucleotide poly(A) mRNA mimic termed A 12 selectively binds to the RNA binding region of eCIRP and inhibits eCIRP binding to its receptor TLR4. Furthermore, in vivo administration of eCIRP induces lung injury in healthy mice and that mouse deficient in CIRP showed protection from inflammation-associated lung injury. We hypothesize that A 12 inhibits systemic inflammation and ALI in HS. To test the impacts of A 12 on systemic and lung inflammation, extent of inflammatory cellular infiltration and resultant lung damage were evaluated in a mouse model of HS. Male mice were subjected to controlled hemorrhage with a mean arterial pressure of 30 mm Hg for 90 min and then resuscitated with Ringer's lactate solution containing phosphate-buffered saline (vehicle) or A 12 at a dose of 4 nmol/g body weight (treatment). The infusion volume was twice that of the shed blood. At 4 h after resuscitation, mice were euthanized, and blood and lung tissues were harvested. Blood and tissue markers of inflammation and injury were evaluated. Serum markers of injury (lactate dehydrogenase, alanine transaminase, and blood urea nitrogen) and inflammation (TNF-α, IL-6) were increased after HS and A 12 treatment significantly decreased their levels. A 12 treatment also decreased lung levels of TNF-α, MIP-2, and KC mRNA expressions. Lung histological injury score, neutrophil infiltration (Ly6G staining and myeloperoxidase activity), and lung apoptosis were significantly attenuated after A 12 treatment. Our study suggests that the capacity of A 12 in attenuating HS-induced ALI and may provide novel perspectives in developing efficacious pharmaceutics for improving hemorrhage prognosis.

The protective effect of Sanggenol L against DMBA-induced hamster buccal pouch carcinogenesis induces apoptosis and inhibits cell proliferative signalling pathway.

BACKGROUND: Oral squamous cell carcinoma (OSCC) has a poor prognosis when treated with surgery and chemotherapy. Therefore, a new therapy and preventative strategy for OSCC and its underlying mechanisms are desperately needed. The purpose of this study was to examine the chemopreventive effects of sanggenol L on oral squamous cell carcinoma (OSCC). The research focused on molecular signalling pathways in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. AIM: The purpose of this study was to look at the biochemical and chemopreventive effects of sanggenol L on 7,12-dimethylbenz(a)anthracene (DMBA)-induced HBP (hamster buccal pouch) carcinogenesis via cell proliferation and the apoptotic pathway. METHODS: After developing squamous cell carcinoma, oral tumours continued to progress leftward into the pouch 3 times per week for 10 weeks while being exposed to 0.5 % reactive DMBA three times per week. Tumour growth was caused by biochemical abnormalities that induced inflammation, increased cell proliferation, and decreased apoptosis. RESULTS: Oral sanggenol L (10 mg/kg bw) supplementation with cancer-induced model DMBA-painted hamsters prevented tumour occurrences, improved biochemistry, inhibited inflammatory markers, decreased cell proliferation marker expression of tumour necrosis factor-alpha (TNF- α), nuclear factor (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and induced apoptosis. CONCLUSION: Sanggenol L could be developed into a new medicine for the treatment of oral carcinogenesis.

Development of human cell-expressed tag-free rhMFG-E8 as a radiation mitigator and a therapeutic for acute kidney injury.

Background: Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. E. coli-expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and possible antigenicity, E. coli-expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 can be developed as a safe and effective novel biologic to treat inflammatory diseases such as radiation injury and acute kidney injury (AKI). Methods: We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity in vitro. We then determined its efficacy in vivo utilizing two experimental rodent models of organ injury: partial body irradiation (PBI) and ischemia/reperfusion-induced AKI. Results: HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE analysis and mass spectrometry. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of E. coli-expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with an adequate half-life for therapeutic applications. In the PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 was 1.073. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI. In the model of AKI, tag-free rhMFG-E8 treatment significantly attenuated kidney injury and inflammation, and improved the 10-day survival. Conclusion: Our new human cell-expressed tag-free rhMFG-E8 can be further developed as a safe and effective therapy to treat victims of severe acute radiation injury and patients with acute kidney injury.

The protective effect of H151, a novel STING inhibitor, in renal ischemia-reperfusion-induced acute kidney injury.

Renal ischemia-reperfusion (RIR)-induced acute kidney injury (AKI) is a common renal functional disorder with high morbidity and mortality. Stimulator of interferon (IFN) genes (STING) is the cytosolic DNA-activated signaling pathway that mediates inflammation and injury. Our recent study showed that extracellular cold-inducible RNA-binding protein (eCIRP), a newly identified damage-associated molecular pattern, activates STING and exacerbates hemorrhagic shock. H151 is a small molecule that selectively binds to STING and inhibits STING-mediated activity. We hypothesized that H151 attenuates eCIRP-induced STING activation in vitro and inhibits RIR-induced AKI in vivo. In vitro, renal tubular epithelial cells incubated with eCIRP showed increased levels of IFN-ß, STING pathway downstream cytokine, IL-6, tumor necrosis factor-α, and neutrophil gelatinase-associated lipocalin, whereas coincubation with eCIRP and H151 diminished those increases in a dose-dependent manner. In vivo, 24 h after bilateral renal ischemia-reperfusion, glomerular filtration rate was decreased in RIR-vehicle-treated mice, whereas glomerular filtration rate was unchanged in RIR-H151-treated mice. In contrast to sham, serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin were increased in RIR-vehicle, but in RIR-H151, these levels were significantly decreased from RIR-vehicle. In contrast to sham, kidney IFN-ß mRNA, histological injury score, and TUNEL staining were also increased in RIR-vehicle, but in RIR-H151, these levels were significantly decreased from RIR-vehicle. Importantly, in contrast to sham, in a 10-day survival study, survival decreased to 25% in RIR-vehicle, but RIR-H151 had a survival of 63%. In conclusion, H151 inhibits eCIRP-induced STING activation in renal tubular epithelial cells. Therefore, STING inhibition by H151 can be a promising therapeutic intervention for RIR-induced AKI.NEW & NOTEWORTHY Renal ischemia-reperfusion (RIR)-induced acute kidney injury (AKI) is a common renal functional disorder with a high morbidity and mortality rate. Stimulator of interferon genes (STING) is the cytosolic DNA-activated signaling pathway responsible for mediating inflammation and injury. Extracellular cold-inducible RNA-binding protein (eCIRP) activates STING and exacerbates hemorrhagic shock. H151, a novel STING inhibitor, attenuated eCIRP-induced STING activation in vitro and inhibited RIR-induced AKI. H151 shows promise as a therapeutic intervention for RIR-induced AKI.

A pharmacokinetic analysis of amisulpride in adult Chinese patients with schizophrenia: Impact of creatinine clearance.

OBJECTIVE: To develop a stable population pharmacokinetic (PPK) model of amisulpride and to investigate the effects of covariates on the pharmacokinetic parameters in adult Chinese patients with schizophrenia. MATERIALS AND METHODS: This retrospective study was carried out using 168 serum samples from 88 patients collected during routine clinical monitoring. Covariates recorded included demographic parameters (gender, age, weight), clinical parameters (serum creatinine, creatinine clearance), and intake of co-medications. The amisulpride PPK model was established using a nonlinear mixed effects modeling (NONMEM) approach. Goodness-of-fit (GOF) plots, bootstrap validation (1,000 runs), and normalized prediction distribution error (NPDE) were used in the evaluation of the final model. RESULTS: A one-compartment model with first-order absorption and elimination was developed. The population estimates for apparent clearance (CL/F) and apparent volume of distribution (V/F) were 32.6 L/h and 391 L, respectively. Estimated creatinine clearance (eCLcr) was a significant covariate for CL/F. The established model was: CL/F = 32.6 × (eCLcr/114.3)0.485 (L/h). The stability of the model was confirmed using GOF plots, bootstrap, and NPDE. CONCLUSION: Creatinine clearance is a major covariate which is positively correlated with CL/F. Therefore, additional dose adjustments of amisulpride may be required on the basis of eCLcr. An ethnic difference may exist in the pharmacokinetics of amisulpride, but further research is needed in order to confirm this possibility. The PPK model of amisulpride for adult Chinese schizophrenic patients established here using NONMEM, is potentially an important tool for individualizing drug dosage and therapeutic drug monitoring.

High-Performance Bifunctional Porous Iron-Rich Phosphide/Nickel Nitride Heterostructures for Alkaline Seawater Splitting.

Seawater is the most abundant natural water resource in the world, which is an inexhaustible and low-cost feedstock for hydrogen production by alkaline water electrolysis. It is appearling to develop robust and stable electrocatalysts for alkaline seawater electrolysis. However, the development of seawater electrolysis is seriously impeded by anodic chloride corrosion and chlorine evolution reaction, and few non-noble electrocatalysts show prominent catalytic performance and excellent durability. Here, a heterogeneous electrocatalyst constructed by in situ growing highly dispersed iron-rich bimetallic phosphide nanoparticles on metallic Ni3 N (Fe2-2 x Co2 x P/Ni3 N), which exhibits outstanding bifunctional catalytic activities for alkaline seawater splitting, is reported. The optimal (Fe0.74 Co0.26 )2 P/Ni3 N and Fe2 P/Ni3 N electrocatalysts demand only 113 and 212 mV to afford 100 mA cm-2 for hydrogen and oxygen evolution reactions (HER and OER) in 1 m KOH, respectively, thus substantially expediting overall water/seawater electrolysis at 100 mA cm-2 with 1.592/1.645 V. Particularly, Fe2 P/Ni3 N displays an unprecedented overpotential of 302 mV at 500 mA cm-2 , which represents the best alkaline seawater oxygen evolution activity among the ever-reported non-noble electrocatalysts; and thus substantially expedites overall water/seawater splitting at 500 mA cm-2 with 1.701/1.768 V, surpassing most of the reported non-noble lectrocatalysts. This work provides a new approach for developing high-performance electrocatalysts for seawater splitting.

Engineering Multilevel Collaborative Catalytic Interfaces with Multifunctional Iron Sites Enabling High-Performance Real Seawater Splitting.

Given the abundant reserves of seawater and the scarcity of freshwater, real seawater electrolysis is a more economically appealing technology for hydrogen production relative to orthodox freshwater electrolysis. However, this technology is greatly precluded by the undesirable chlorine oxidation reaction and severe chloride corrosion at the anode, further restricting the catalytic efficiency of overall seawater splitting. Herein, a feasible strategy by engineering multifunctional collaborative catalytic interfaces is reported to develop porous metal nitride/phosphide heterostructure arrays anchoring on conductive Ni2P surfaces with affluent iron sites. Collaborative catalytic interfaces among iron phosphide, bimetallic nitride, and porous Ni2P supports play a positive role in improving water adsorption/dissociation and hydrogen adsorption behaviors of active Fe sites evidenced by theoretical calculations for hydrogen evolution reactions, and enhancing oxygenated species adsorption and nitrate-rich passivating layers resistant to chloride corrosion for oxygen evolution reaction, thus cooperatively propelling high-performance bifunctional seawater splitting. The resultant material Fe2P/Ni1.5Co1.5N/Ni2P performs excellently as a self-standing bifunctional catalyst for alkaline seawater splitting. It requires extremely low cell voltages of 1.624 and 1.742 V to afford current densities of 100 and 500 mA/cm2 in 1 M KOH seawater electrolytes, respectively, along with superior long-term stability, outperforming nearly all the ever-reported non-noble bifunctional electrocatalysts and benchmark Pt/IrO2 coupled electrodes for freshwater/seawater electrolysis. This work presents an effective strategy for greatly enhancing the catalytic efficiency of non-noble catalysts toward green hydrogen production from seawater electrolysis.

Extracellular CIRP induces acute kidney injury via endothelial TREM-1.

Introduction: Acute kidney injury is associated with elevated serum levels of extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern released during ischemia/reperfusion injury, hemorrhagic shock, and sepsis. It is unknown if circulating eCIRP and eCIRP-induced activation of receptor triggering receptor expressed on myeloid cells-1 (TREM-1), expressed on endothelial cells, play an important role in the pathogenesis of AKI. Methods: Male B6 wild-type (WT) and TREM-1-/- mice were subjected to intravenous injection of recombinant murine (rm) CIRP. Serum, urine, and renal tissue were collected 6 h later for analysis. Additionally, primary human renal glomerular endothelial cells (HRGEC) were stimulated in vitro with rmCIRP after pretreatment with M3, a novel inhibitory peptide of TREM-1, or vehicle. Supernatants and cells were collected 20 h after stimulation. Results: After injection with rmCIRP, WT mice had a significant increase in serum levels of BUN, creatinine, and NGAL compared to control. Additionally, NGAL was significantly increased in the urine of rmCIRP-injected mice, suggesting that circulating eCIRP can directly induce AKI. The levels of TREM-1 mRNA in the kidneys, as well as soluble (s) TREM-1 released into the serum and urine, were significantly increased in rmCIRP-injected mice. TREM-1-/- mice injected with rmCIRP had attenuated AKI, indicated by significantly decreased serum BUN, creatinine, and NGAL, and renal mRNA expression of NGAL and KIM-1 compared to WT mice. TREM-1-/- mice also had attenuated endothelial activation, with decreased mRNA and protein expression of ICAM-1 in renal tissue. HRGEC stimulated with rmCIRP in vitro had significant increases in cytokine production and sTREM-1 release, which was attenuated in cells treated with M3. Conclusion: Activation of renal TREM-1 with circulating eCIRP is sufficient to cause AKI. Elevated levels of eCIRP may be critical for the development of AKI under conditions such as ischemia/reperfusion injury, hemorrhagic shock, and sepsis. Mice deficient in the TREM-1 receptor have attenuated AKI and reduced endothelial cell activation after injection of rmCIRP. TREM-1 inhibition with M3 attenuates HRGEC activation after eCIRP stimulation. Targeting eCIRP activation of TREM-1 may provide a novel and effective treatment for AKI.

The enhanced genomic 6 mA metabolism contributes to the proliferation and migration of TSCC cells.

In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N6-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.

Synergistic Silencing of Skp2 by siRNA Self-Assembled Nanoparticles as a Therapeutic Strategy for Advanced Prostate Cancer.

Advanced prostate cancer, harboring multiple mutations of tumor suppressor genes, is refractory to conventional therapies. Knockout of the Skp2 gene blocks pRb/p53 doubly deficient prostate cancer in mice, which inspired the authors to develop an approach for delivering siRNA that would efficiently silence Skp2 (siSkp2) in vivo. Here, a facile strategy is reported to directly assemble siSkp2 with the natural compound quercetin (Que) into supramolecular nanoparticles (NPs). This carrier-free siSkp2 delivery system could effectively protect siSkp2 from degradation in serum and enhance its cellular internalization. Furthermore, the siSkp2/Que NPs exhibit synergistic effects in Skp2 silencing, because they can degrade the mRNA and protein of Skp2 simultaneously. Indeed, siSkp2/Que NPs remarkably diminish the Skp2 abundance and further inhibit the proliferation and migration of TMU cells (RB1/TP53/KRAS triple mutations) in vitro. The in vivo results further show that i.v. administration of siSkp2/Que NPs efficiently accumulates in tumor sites and strongly inhibits the growth of TMU tumors in nude mice. Importantly, the siSkp2/Que NPs do not induce any abnormality in the treated mice, which suggests satisfactory biocompatibility. Collectively, this study describes a tractable siRNA self-assembled strategy for Skp2 silencing, which might be a promising nanodrug to cure multitherapy-resistant advanced prostate cancer.

Rh(iii)-catalyzed regioselective C-H activation dialkenylation/annulation cascade for rapid access to 6H-isoindolo[2,1-a]indole.

6H-isoindolo[2,1-a]indoles were accessed via a Rh(iii)-catalyzed N-H free indole directed C-H activation dialkenylation/annulation cascade in moderate to excellent yields. This protocol also features: reaction procedures that are insensitive to air and moisture, excellent regioselectivity and good functional group tolerance.

Characterization of antiapoptotic microRNAs in primary Sjögren's syndrome.

During the development of primary Sjögren's syndrome (pSS), aberrant expression of autoantigen is a hallmark event. To explore the regulation of autoantigen tripartite motif containing 21 (Ro/SSA, TRIM21), microRNA profiling was performed in our previous study. In which, two TRIM21-targeting microRNAs were identified, namely miR-1207-5p and miR-4695-3p. To further pursue their roles in the development of pSS, assays were performed with cultured human submandibular gland (HSG) cells, and salivary gland tissues. Results showed that transfection of miR-1207-5p or miR-4695-3p mimics down-regulated not only the expression of TRIM21, but also the levels of pro-apoptotic genes B cell lymphoma 2 associated X (BAX), Caspase 9 (CASP-9) and Caspase 8 (CASP-8). This finally led to antiapoptotic phenotypes in HSG cells. Consistent with the antiapoptotic activity, transfection of microRNA inhibitors up-regulated the expression of TRIM21 and led to a pro-apoptotic phenotype. These therefore propose miR-1207-5p and miR-4695-3p as two antiapoptotic microRNAs functioning through apoptosis pathway. Supporting this speculation, assays performed with salivary gland tissues revealed down-regulation of miR-1207-5p and miR-4695-3p, as well as up-regulation of TRIM21 and pro-apoptotic CASP-8 gene in pSS samples. SIGNIFICANCE OF THE STUDY: For pSS patients, apoptosis of acinar and ductal epithelial cells has been proposed to be a potential mechanism that impairs the secretion of salivary glands. In our study, two autoantigen-targeting microRNAs were characterized as antiapoptotic microRNAs functioning through apoptosis pathway, which may be potential targets for the treatment of pSS.

Cu/ZnSOD always responded stronger and rapider than MnSOD in Lymantria dispar larvae under the avermectin stress.

Metalloenzyme SODs play important roles in insects dealing with environmental stress. Here, we cloned the Cu/ZnSOD (LdCZS) and MnSOD (LdMS) mRNA of Lymantria dispar by rapid amplification of cDNA ends (RACE). Afterwards their expression patterns were detected by quantitative real-time polymerase chain reaction (qPCR) after bioinformatic analysis. We found that both LdCZS and LdMS were widely detected in all gypsy moth larvae and all five tissues that we analyzed, and both of them were up-regulated after larvae were fed with avermectin of sublethal concentration and LC10. The LdCZS expression value are always higher than LdMS after treating with avermectin of sublethal concentrations. In addition, temporal expression profile in avermectin treated larvae showed that LdCZS expressed highest at 2nd hour, and LdMS expressed highest at 6th hour. The cuticulas transcribed LdCZS and LdMS significantly higher than heads, fat bodies, Malpighian tubes, and midguts after spraying avermectin of sublethal concentration. These results suggested that both Cu/ZnSOD and MnSOD are important antioxidant enzymes in L. dispar defensing against pesticide stress, and LdCZS always responded rapider and stronger than LdMS.

Osteogenic capacity and cytotherapeutic potential of periodontal ligament cells for periodontal regeneration in vitro and in vivo.

BACKGROUND: The periodontal ligament cells (PDLCs) contain heterogeneous cell populations and possess stem-cell-like properties. PDLCs have attracted considerable attention as an option for periodontal regeneration. However, the osteogenic differentiation of PDLCs remains obscure owing to variable osteo-inductive methods and whether PDLCs could be directly used for periodontal regeneration without stem cell enrichment is uncertain. The aim of the present study was to clarify the osteogenic differentiation capacity of PDLCs and test PDLCs as an alternative to stem cells for periodontal regeneration. METHODS: We tested the performance of human PDLCs in osteo-inductive culture and transplantation in vivo while taking human bone marrow derived mesenchymal stem cells (hMSCs) as positive control. Proliferation of PDLCs and hMSCs in osteo-inductive condition were examined by MTT assay and colony formation assay. The osteogenic differentiations of PDLCs and hMSCs were assessed by Alkaline phosphatase (ALP) activity measurement, von Kossa staining, Alizarin red S staining and quantitative RT-PCR of osteogenic marker gene including RUNX2, ALP, OCN, Col I, BSP, OPN. We transplanted osteo-inductive PDLCs and hMSCs with hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds to immunodeficient mice to explore their biological behaviors in vivo by histological staining and immunohistochemical evaluation. RESULTS: After 14 days of osteo-induction, PDLCs exhibited significantly higher proliferation rate but lower colony-forming ability comparing with hMSCs. PDLCs demonstrated lower ALP activity and generated fewer mineralized nodules than hMSCs. PDLCs showed overall up-regulated expression of RUNX2, ALP, OCN, Col I, BSP, OPN after osteo-induction. Col I level of PDLCs in osteo-inductive group was significantly higher while RUNX2, ALP, OCN were lower than that of hMSCs. Massive fiber bundles were produced linking or circling the scaffold while the bone-like structures were limited in the PDLCs-loaded HA/TCP samples. The fiber bundles displayed strong positive Col I, but weak OCN and OPN staining. The in vivo results were consistent with the in vitro data, which confirmed strong collagen forming ability and considerable osteogenic potential of PDLCs. CONCLUSION: It is encouraging to find that PDLCs exhibit higher proliferation, stronger collagen fiber formation capacity, but lower osteogenic differentiation ability in comparison with hMSCs. This characteristic is essential for the successful periodontal reconstruction which is based on the synchronization of fiber formation and bone deposition. Moreover, PDLCs have advantages such as good accessibility, abundant source, vigorous proliferation and evident osteogenic differentiation capacity when triggered properly. They can independently form PDL-like structure in vivo without specific stem cell enrichment procedure. The application of PDLCs may offer a novel cytotherapeutic option for future clinical periodontal reconstruction.

Cold-inducible RNA-binding protein-derived peptide C23 attenuates inflammation and tissue injury in a murine model of intestinal ischemia-reperfusion.

BACKGROUND: Cold-inducible RNA-binding protein is a novel damage-associated molecular pattern that causes inflammation. C23, a short peptide derived from cold-inducible RNA-binding protein, has been found to have efficacy in blocking cold-inducible RNA-binding protein's activity. We hypothesized that C23 reduces inflammation and tissue injury induced by intestinal ischemia-reperfusion. METHODS: Male C57BL/6 mice were subjected to 60 minutes of intestinal ischemia by clamping the superior mesenteric artery. Immediately after reperfusion, either normal saline (vehicle) or C23 peptide (8 mg/kg body weight) was injected intraperitoneally. Four hours after reperfusion, blood, intestinal, and lung tissues were collected for analysis of inflammatory and tissue injury parameters. RESULTS: Cold-inducible RNA-binding protein levels in the intestinal tissues were significantly increased following intestinal ischemia-reperfusion. Histologic examination of the intestine revealed a significant reduction in injury score in the C23 group by 48% as compared with the vehicles after intestinal ischemia-reperfusion. The serum levels of lactate dehydrogenase and aspartate aminotransferase were increased in animals that underwent vehicle-treated intestinal ischemia-reperfusion, whereas C23-treated animals exhibited significant reductions by 48% and 53%, respectively. The serum and intestinal tissue levels of tumor necrosis factor α were elevated in vehicle-treated intestinal ischemia-reperfusion mice but decreased by 72% and 69%, respectively, in C23-treated mice. Interleukin-6 mRNA levels in the lungs were reduced by 86% in the C23-treated group in comparison to the vehicle-treated group after intestinal ischemia-reperfusion. Expression of macrophage inflammatory protein 2 and level of myeloperoxidase activity in the lungs were dramatically increased after intestinal ischemia-reperfusion and significantly reduced by 91% and 25%, respectively, in the C23-treated group. CONCLUSION: C23 has potential to be developed into a possible therapy for reperfusion injury after mesenteric ischemia and reperfusion.

A cold-inducible RNA-binding protein (CIRP)-derived peptide attenuates inflammation and organ injury in septic mice.

Cold-inducible RNA-binding protein (CIRP) is a novel sepsis inflammatory mediator and C23 is a putative CIRP competitive inhibitor. Therefore, we hypothesized that C23 can ameliorate sepsis-associated injury to the lungs and kidneys. First, we confirmed that C23 dose-dependently inhibited TNF-α release, IκBα degradation, and NF-κB nuclear translocation in macrophages stimulated with CIRP. Next, we observed that male C57BL/6 mice treated with C23 (8 mg/kg BW) at 2 h after cecal ligation and puncture (CLP) had lower serum levels of LDH, ALT, IL-6, TNF-α, and IL-1ß (reduced by ≥39%) at 20 h after CLP compared with mice treated with vehicle. C23-treated mice also had improved lung histology, less TUNEL-positive cells, lower serum levels of creatinine (34%) and BUN (26%), and lower kidney expression of NGAL (50%) and KIM-1 (86%). C23-treated mice also had reduced lung and kidney levels of IL-6, TNF-α, and IL-1ß. E-selectin and ICAM-1 mRNA was significantly lower in C23-treated mice. The 10-day survival after CLP of vehicle-treated mice was 55%, while that of C23-treated mice was 85%. In summary, C23 decreased systemic, lung, and kidney injury and inflammation, and improved the survival rate after CLP, suggesting that it may be developed as a new treatment for sepsis.

The Protective Effect of A Short Peptide Derived From Cold-Inducible RNA-Binding Protein in Renal Ischemia-Reperfusion Injury.

Extracellular cold-inducible RNA-binding protein (CIRP) functions as damage-associated molecular pattern and has been demonstrated to be responsible in part for the damage occurring after renal ischemia-reperfusion (I/R). A short peptide derived from CIRP, named C23, binds to myeloid differentiation factor 2, a Toll-like receptor 4 coreceptor. We hypothesize that C23 reduces renal ischemia-reperfusion (RIR) injury by blocking CIRP. We observed that pretreatment with C23 significantly decreased the levels of recombinant mouse CIRP-induced tumor necrosis factor-α (TNF-α) in a dose-dependent fashion in cultured macrophages. C57BL/6 mice were subjected to bilateral renal pedicle clamps for 35 min to induce ischemia, followed by reperfusion for 24 h and harvest of blood and renal tissue. C23 peptide (8 mg/kg) or vehicle was injected intraperitoneally at the beginning of reperfusion. Plasma TNF-α, interleukin 1 beta (IL-1ß), and IL-6 levels were decreased in C23-treated RIR mice as compared with vehicle-treated mice by 74%, 85%, and 68%, respectively. Expressions of TNF-α and keratinocyte chemoattractant in the kidneys from C23-treated mice were decreased by 55% and 60%, respectively. Expression of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in the kidney of C23-treated mice were significantly reduced by 46% and 55%, respectively. Renal tissue histological assessments revealed significant reduction in damage score by 44% in C23-treated mice. Finally, a survival study revealed a significant survival advantage with a 70% survival rate in C23 group vs. 37% in vehicle group. Thus, C23 has potential as a novel therapy for the patients suffering from I/R-induced renal injury.