mmu-lncRNA 121686/hsa-lncRNA 520657 induced by METTL3 drive the progression of AKI by targeting miR-328-5p/HtrA3 signaling axis.

The pathogenesis of acute kidney injury (AKI) is still not fully understood, and effective interventions are lacking. Here, we explored whether methyltransferase 3 (METTL3) was involved in the progression of AKI via regulation of cell death. We reported that PT（proximal tubule）-METTL3-knockout (KO) noticeably suppressed ischemic-induced AKI via inhibition of renal cell apoptosis. Furthermore, we also found that the expression of mmu-long non-coding RNA (lncRNA) 121686 was upregulated in antimycin-treated Boston University mouse proximal tubule (BUMPT) cells and a mouse ischemia-reperfusion (I/R)-induced AKI model. Functionally, mmu-lncRNA 121686 could promote I/R-induced mouse renal cell apoptosis. Mechanistically, mmu-lncRNA 121686 acted as a competing endogenous RNA (ceRNA) to prevent microRNA miR-328-5p-mediated downregulation of high-temperature requirement factor A 3 (Htra3). PT-mmu-lncRNA 121686-KO mice significantly ameliorated the ischemic-induced AKI via the miR-328-5p/HtrA3 axis. In addition, hsa-lncRNA 520657, homologous with lncRNA 121686, sponged miR-328-5p and upregulated Htra3 to promote I/R-induced human renal cell apoptosis. Interestingly, we found that mmu-lncRNA 121686/hsa-lncRNA 520657 upregulation were dependent on METTL3 via N6-methyladenosine (m6A) modification. The mmu-lncRNA 121686/miR-328-5p or hsa-lncRNA 520657/miR-328-5p /HtrA3 axis was induced in vitro by METTL3 overexpression; in contrast, this effect was attenuated by METTL3 small interfering RNA (siRNA). Furthermore, we found that PT-METTL3-KO or METTL3 siRNA significantly suppressed ischemic, septic, and vancomycin-induced AKI via downregulation of the mmu-lncRNA 121686/miR-328-5p/HtrA3 axis. Taken together, our data indicate that the METTL3/mmu-lncRNA 121686/hsa-lncRNA 520657/miR-328-5p/HtrA3 axis potentially acts as a therapeutic target for AKI.

Genetic or siRNA inhibition of MBD2 attenuates the UUO- and I/R-induced renal fibrosis via downregulation of EGR1.

DNA methylation plays a pivotal role in the progression of renal fibrosis. Methyl-CpG-binding domain protein 2 (MBD2), a protein reader of methylation, is involved in the development of acute kidney injury (AKI) caused by vancomycin. However, the role and mechanism of action of MBD2 in renal remain unclear. In this study, MBD2 mediated extracellular matrix (ECM) production induced by TGF-ß1 in Boston University mouse proximal tubule (BUMPT) cells，and upregulated the expression EGR1 to promote ECM production in murine embryonic NIH 3T3 fibroblasts. ChIP analysis demonstrated that MBD2 physically interacted with the promoter region of the CpG islands of EGR1 genes and then activated their expression by inducing hypomethylation of the promoter region. In vivo, PT-MBD2-KO attenuated unilateral ureteral obstruction (UUO)-induced renal tubulointerstitial fibrosis via downregulation of EGR1, which was demonstrated by the downregulation of fibronectin (FN), collagen I and IV, α-SMA, and EGR1. Injection of MBD2-siRNA attenuated the UUO- and I/R-induced renal fibrosis. Those molecular changes were verified by biopsies from patients with obstructive nephropathy (OB). These data collectively demonstrated that inhibition of MBD2 reduces renal fibrosis via downregulating EGR1, which could be a target for treatment of fibrotic kidney disease.

DsbA-L interacts with VDAC1 in mitochondrion-mediated tubular cell apoptosis and contributes to the progression of acute kidney disease.

BACKGROUND: we demonstrated that disulfide-bond A oxidoreductase-like protein (DsbA-L) was involved in the progression of renal fibrosis. However, the precise function of DsbA-L in acute kidney injury (AKI), and the mechanisms involved, have yet to be elucidated. METHODS: We illustrate the DsbA-L interacted with VDAC1 by co-IP (co-immunoprecipitation) in vitro and vivo, and found the interaction parts of them by mutation experiment. The above findings were verified by co-localization of them. In addition, we constructed the two model of PT-DsbA-L and VDAC1 KO mice to verify the function of DsbA-L and VDAC1 in models of VAN, CLP and I/R-induced AKI. FINDINGS: The PT-DsbA-L-KO mice showed amelioration of I/R, VAN-, and CLP-induced AKI progression via the downregulation of VDAC1. Finally, we confirmed these changes in signal molecules by examining in HK-2 cells and kidney biopsies taken from patients with ischemic or acute interstitial nephritis (AIN)-induced AKI. Mechanistically, DsbA-L interacted with amino acids 9-13 and 22-27 of VDAC1 in the mitochondria of BUMPT cells to induce renal cell apoptosis and mitochondrial injury. INTERPRETATION: This work suggested that DsbA-L, located in the proximal tubular cells, drives the progression of AKI, by directly upregulating the levels of VDAC1.Running Title: The role of DsbA-L in AKI FUNDING: National Natural Science Foundation of China, a grant from Key Project of Hunan provincial science and technology innovation, Department of Science and Technology of Hunan Province project of International Cooperation and Exchanges, Changsha Science and Technology Bureau project, Natural Science Foundation of Hunan Province, Fundamental Research Funds for the Central Universities of Central South University, Hunan Provincial Innovation Foundation For Postgraduate China Hunan Provincial Science and Technology Department.

Methyl-CpG-binding domain protein 2 contributes to renal fibrosis through promoting polarized M1 macrophages.

Recent studies reported that Methyl-CpG-binding domain protein 2 (MBD2) promoted M2 macrophages accumulation to increase bleomycin-induced pulmonary fibrosis. However, the role and mechanism of action of MBD2 in macrophages differentiation and renal fibrosis remain largely unknown. In the current study, MBD2 not only promoted the differentiation of resting M0 macrophages to polarized M2 macrophages, but also induced them to polarized M1 macrophages and the transition of M2 to M1 macrophages. ChIP analysis demonstrated that MBD2 physically interacted with the promoter region of the CpG islands of G0S2 genes, and then activated their expression by inducing hypomethylation of the promoter region. Interestingly, the data demonstrated that the role of G0S2 in macrophages differentiation is consistent with MBD2. Furthermore, Co-culture of activated M1 macrophages and murine embryonic NIH 3T3 fibroblasts indicated that MBD2 mediated the M1-induction of ECM production by embryonic NIH 3T3 fibroblasts via promotion of G0S2. In addition, we also found that inhibition of MBD2 suppressed LPS induced the expression of p53 as well as activation and expression of stat3 in RAW264.7 macrophages. In vivo, MBD2 LysMcre attenuated unilateral ureteral obstruction (UUO) and ischemia/reperfusion (I/R)-induced renal fibrosis via downregulation of G0S2, which was demonstrated by the downregulation of fibronectin (FN), collagen I and IV, α-SMA, G0S2. These data collectively demonstrated that MBD2 in macrophages contributed to UUO and I/R-induced renal fibrosis through the upregulation of G0S2, which could be a target for treatment for chronic kidney disease.

Loss of MBD2 ameliorates LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis.

Apoptosis of alveolar epithelial cells is a critical initial link in the pathogenesis of acute lung injury (ALI), recent studies have revealed that Methyl-CpG binding domain protein 2 (MBD2) was involved in the execution of apoptosis, yet its role in ALI remained unclear. In the present study, we aim to explore the role and mechanism of MBD2 in the pathogenesis of ALI. We have found that MBD2 expression, in parallel to apoptosis, increased in alveolar epithelial cells of mice treated with LPS, knockout of MBD2 reduced apoptosis and protected mice from LPS-induced ALI. In MLE-12 cells, a cell line of murine alveolar epithelial cells, LPS induced MBD2 expression and apoptosis in a dose- and time-dependent manner. Knockdown of MBD2 with shRNA alleviated, while overexpression of MBD2 increased LPS-induced apoptosis. Mechanistically, intracellular zinc level decreased when MLE-12 cells were treated with LPS. MBD2 knockdown restored intracellular zinc level after LPS treatment, and MBD2 overexpression further aggravated LPS-induced intracellular zinc loss. Metal transcription factor 1 (MTF1) is a critical transcription factor in charge of intracellular zinc efflux. LPS treatment induced MTF1 expression both in vivo and in vitro. Inhibition of MTF1 reduced LPS-induced apoptosis in MLE-12 cells. MBD2 could bind to the promoter region of MTF1 and promote MTF1 expression. Collectively, these data indicated that loss of MBD2-ameliorated LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis by upregulating MTF1.

A Comparative Transcriptional Landscape of Two Castor Cultivars Obtained by Single-Molecule Sequencing Comparative Analysis.

Background and Objectives: Castor (Ricinus communis L.) is an important non-edible oilseed crop. Lm-type female strains and normal amphiprotic strains are important castor cultivars, and are mainly different in their inflorescence structures and leaf shapes. To better understand the mechanisms underlying these differences at the molecular level, we performed a comparative transcriptional analysis. Materials and Methods: Full-length transcriptome sequencing and short-read RNA sequencing were employed. Results: A total of 76,068 and 44,223 non-redundant transcripts were obtained from high-quality transcripts of Lm-type female strains and normal amphiprotic strains, respectively. In Lm-type female strains and normal amphiprotic strains, 51,613 and 20,152 alternative splicing events were found, respectively. There were 13,239 transcription factors identified from the full-length transcriptomes. Comparative analysis showed a great variety of gene expression of common and unique transcription factors between the two cultivars. Meanwhile, a functional analysis of the isoforms was conducted. The full-length sequences were used as a reference genome, and a short-read RNA sequencing analysis was performed to conduct differential gene analysis. Furthermore, the function of DEGs were performed to annotation analysis. Conclusion: The results revealed considerable differences and expression diversity between the two cultivars, well beyond what was reported in previous studies and likely reflecting the differences in architecture between these two cultivars.

Lysophosphatidic Acid May Be a Novel Biomarker for Early Acute Aortic Dissection.

Background: Misdiagnosis and delayed diagnosis of acute aortic dissection (AAD) significantly increase mortality. Lysophosphatidic acid (LPA) is a biomarker related to coagulation cascade and cardiovascular-injury. The extent of LPA elevation in AAD and whether it can discriminate sudden-onset of acute chest pain are currently unclear. Methods: We measured the plasma concentration of LPA in a cohort of 174 patients with suspected AAD chest pain and 30 healthy participants. Measures to discriminate AAD from other acute-onset thoracalgia were compared and calculated. Results: LPA was significantly higher in AAD than in the AMI, PE, and the healthy (344.69 ± 59.99 vs. 286.79 ± 43.01 vs. 286.61 ± 43.32 vs. 96.08 ± 11.93, P < 0.01) within 48 h of symptom onset. LPA level peaked at 12 h after symptom onset, then gradually decreased from 12 to 48 h in AAD. LPA had an AUC of 0.85 (0.80-0.90), diagnosis threshold of 298.98 mg/dl, a sensitivity of 0.81, specificity of 0.77, and the negative predictive value of 0.85. The ROC curve of LPA is better than D-dimer (P = 0.041, Delong test). The decision curve showed that LPA had excellent standardized net benefits. Conclusion: LPA showed superior overall diagnostic performance to D-dimer in early AAD diagnosis may be a potential biomarker, but additional studies are needed to determine the rapid and cost-effective diagnostic tests in the emergency department.

Multiple soft tissue inflammation as the first symptom in lymphoma: A case report. / 以多发软组织炎症为首发表现的淋巴瘤1例.

The clinical manifestations of lymphoma are various, and the atypical manifestations lead to difficult diagnosis and misdiagnosis. A 29-year-old male patient was admitted to the hospital due to the symptoms of "three months of left ear purulence, 20 days of right eye swelling and fever". He was diagnosed as "multiple soft tissue infection" in the other hospital. The patient was treated with cefmetazole for anti-infection and dexamethasone for anti-inflammation. His condition has not improved. After being transferred to our hospital, he underwent multiple soft tissue biopsies and was diagnosed as T-cell non-Hodgkin's lymphoma. He was treated with oxaliplatin combined with gemcitabine. The experience of this case suggests that young people should be highly aware of the possibility of lymphoma when they have unexplained infection. Multiple tissue biopsies are very important for clear diagnosis.

DsbA-L mediated renal tubulointerstitial fibrosis in UUO mice.

Recent studies have reported that upregulation of disulfide-bond A oxidoreductase-like protein (DsbA-L) prevented lipid-induced renal injury in diabetic nephropathy (DN). However, the role and regulation of proximal tubular DsbA-L for renal tubulointerstitial fibrosis (TIF) remains unclear. In current study, we found that a proximal tubules-specific DsbA-L knockout mouse (PT-DsbA-L-KO) attenuated UUO-induced TIF, renal cell apoptosis and inflammation. Mechanistically, the DsbA-L interacted with Hsp90 in mitochondria of BUMPT cells which activated the signaling of Smad3 and p53 to produce connective tissue growth factor (CTGF) and then resulted in accumulation of ECM of BUMPT cells and mouse kidney fibroblasts. In addition, the progression of TIF caused by UUO, ischemic/reperfusion (I/R), aristolochic acid, and repeated acute low-dose cisplatin was also alleviated in PT-DsbA-L-KO mice via the activation of Hsp90 /Smad3 and p53/CTGF axis. Finally, the above molecular changes were verified in the kidney biopsies from patients with obstructive nephropathy (Ob). Together, these results suggest that DsbA-L in proximal tubular cells promotes TIF via activation of the Hsp90 /Smad3 and p53/CTGF axis.

Molecular Control of Follicular Helper T cell Development and Differentiation.

Follicular helper T cells (Tfh) are specialized helper T cells that are predominantly located in germinal centers and provide help to B cells. The development and differentiation of Tfh cells has been shown to be regulated by transcription factors, such as B-cell lymphoma 6 protein (Bcl-6), signal transducer and activator of transcription 3 (STAT3) and B lymphocyte-induced maturation protein-1 (Blimp-1). In addition, cytokines, including IL-21, have been found to be important for Tfh cell development. Moreover, several epigenetic modifications have also been reported to be involved in the determination of Tfh cell fate. The regulatory network is complicated, and the number of novel molecules demonstrated to control the fate of Tfh cells is increasing. Therefore, this review aims to summarize the current knowledge regarding the molecular regulation of Tfh cell development and differentiation at the protein level and at the epigenetic level to elucidate Tfh cell biology and provide potential targets for clinical interventions in the future.

Paclitaxel attenuates renal interstitial fibroblast activation and interstitial fibrosis by inhibiting STAT3 signaling.

Recent studies have demonstrated that paclitaxel might inhibit renal fibrosis. However, the underlying molecular mechanism remains unclear. In this study, we hypothesized that low-dose paclitaxel may block the STAT3 (signal transducer and activator of transcription 3) signaling to attenuate fibrosis in a mouse model with unilateral ureteral obstruction. Both NRK-49F cells and mice with unilateral ureteral obstruction were treated with paclitaxel. The results showed that paclitaxel treatment resulted in a dose- and time-dependent decrease in tyrosine-phosphorylated STAT3, and inhibited the expression of fibronectin, alpha-smooth muscle actin (α-SMA), and collagen I in cultured NRK-49F cells. S3I-201, an STAT3 inhibitor, also suppressed the expression of fibronectin, α-SMA, and collagen I in cultured NRK-49F cells. Mechanistically, paclitaxel treatment blocked the STAT3 activity by disrupting the association of STAT3 with tubulin and inhibiting STAT3 nucleus translocation. Furthermore, paclitaxel also ameliorated renal fibrosis by down-regulating the expression of fibronectin, α-SMA, and collagen I, and suppressed the infiltration of macrophages and production of TNF-α, IL-1ß, TGF-ß, and ICAM-1 (intercellular adhesion molecule 1) by inhibition of STAT3 activity in obstructive nephropathy. These results suggest that paclitaxel may block the STAT3 activity by disrupting the association of STAT3 with tubulin and inhibiting STAT3 nucleus translocation, consequently leading to the suppression of renal interstitial fibroblast activation and the development of renal fibrosis, and inhibition of proinflammatory cytokine production.

[Protection of noninvasive limb ischemic preconditioning on myocardium in patients undergoing heart valve surgery under cardiopulmonary bypass].

OBJECTIVE: To determine the mechanism of protective effect of noninvasive limb ischemic preconditioning (NIPC) on myocardium of patients with rheumatic heart disease undergoing heart valve surgery under cardiopulmonary bypass (CPB). METHODS: A total of 32 patients with rheumatic heart disease undergoing heart valve surgeries under CPB were randomly divided into 2 groups: a control group(n=16)and an NIPC group(n=16).Tourniguet was used for each patient in the NIPC group around both the upper extremities in turn, inflated for 8 min and deflated for 5 min for 3 cycles. After the anesthesia, the remaining procedures were the same as in the control group. Blood samples were collected from the central vein after the induction of anesthesia (T(1)), 5 min before aortic clamp (T(2)),30 min after aortic opening (T(3)), 6 h after the operation (T(4)), and 24 h after the operation (T(5)) to measure the concentration of cardiac troponin I and creatine kinase MB in the plasma and CGRP and ET-1 in the serum. Pathologic change of the right auricle of the heart tissue during the superior vena cave intubation and extubation was detected. RESULTS: The content of cardiac troponin I and creatine kinase MB at T(4) and T(5) in the 2 groups was higher than that of other time points in the same group, and it reached the peak at T(5). Comparison of the content of cardiac troponin I and creatine kinase MB at T(4) and T(5) in the 2 groups showed significant difference, and that of the NIPC group was lower than the control group(P<0.05).CGRP and ET-1 contents reached the peak at T(2) in the NIPC group and at T(3) in the control group, but the peak of CGRP in the NIPC group was higher than that in the control group(P<0.01).The peak of ET-1 content in the NIPC group was lower than that in the control group(P<0.01). After the CPB, myocardial and mitochondrion impairment was lighter in the NIPC group than in the control group. CONCLUSION: Noninvasive limb ischemic preconditioning can protect the myocardium through increasing CGRP, inhibiting ET-1, and advancing the peak of CGRP and ET-1.

Effect of morphine preconditioning on mitochondrial permeability transition pore after myocardial ischemia-reperfusion injury in rats.

OBJECTIVE: To investigate the effect of morphine preconditioning on mitochondrial permeability transition pore (MPTP) and its protective mechanism after myocardial ischemia-reperfusion injury. METHODS: A rat model of ischemia-reperfusion injury was established. Forty rats were injected with 2-(3)[H] DOG and then divided into 4 groups randomly: a sham operation (S) group, an ischemia-reperfusion injury (IR) group, a morphine preconditioning (Mp+IR) group, and a cyclosporine A preconditioning (CsA+IR) group. We monitored the concentrations of serum creatine kinase-Mb (CK-Mb) and cardiac troponin I (cTnI), and measured myocardial mitochondrial 2-(3)[H] DOG, cytochrome c content, Ca(2+) concentration ([Ca(2+)]m), the velocity of Ca(2+) intake and reaction half time of mitochondrial permeability transition pore (MPTP t(1/2)) in the 4 groups. RESULTS: The concentrations of serum CK-Mb and cTnI decreased more in the Mp+IR group and the CsA+IR group than those of the IR group. The concentrations of 2-(3)[H]DOG and [Ca(2+)]m in the IR group were evidently higher but the level of cytochrome c was lower than those of the sham operation group. The concentrations of 2-(3)[H] DOG and [Ca(2+)]m in the Mp+IR group decreased whereas the concentration of cytochrome c increased compared with those in the IR group. Mitochondrial 2-(3)[H]DOG content was positively correlated with the concentration of calcium (r=0.797, P<0.01). The 2-(3)[H]DOG and [Ca(2+)]m content were negatively correlated with cytochrome c in the IR group (r=-0.805 and r=-0.648, respectively, P<0.01). MPTP t(1/2) in the IR group was shortened evidently, and that in the Mp+IR and CsA+IR group was significantly lengthened. CONCLUSION: Morphine preconditioning may have myocardial protective effect through unburdening the calcium overload and lengthening the MPTP t(1/2).

[Effect of depsides salts from salvia miltiorrhiza on functions of platelet and vascular endothelial cell of patients with chronic obstructive pulmonary disease in acute exacerbation stage].

OBJECTIVE: To investigate the influence of depsides salts from salvia miltiorrhiza (DSM) on the functions of platelet and vascular endothelial cell in severe chronic obstructive pulmonary diseases (COPD) patients with acute exacerbation. METHODS: Forty patients with severe COPD in acute exacerbation stage were randomly divided into two groups: conventional treatment (CT) group and DSM treatment (DSM) group, each consisting of 20 cases, and 20 healthy adults served as control group. All COPD patients were given conventional treatment, while for the patients in DSM group 0.2 g depsides DSM was given through intravenous drip everyday in addition for 2 weeks. The levels of the plasma platelet membrane glycoprotein 140 (GMP140) and von Willebrand factor (vMF) in the blood samples were determined with enzyme linked immunosorbent assay (ELISA) and the level of CD62P/CD61 with flow cytometry (FCM). RESULTS: The level of GMP140 [CT group: (17.51+/-2.75) microg/L, DSM group: (16.94+/-2.57) microg/L], vMF [CT group: (13.64+/-2.37) microg/L, DSM group: (14.14+/-2.17) microg/L] and CD62P/CD61 [CT group: (20.24+/-2.64)% , DSM group: (19.54+/-2.69)%] were elevated significantly in severe COPD patients with acute exacerbation compared to the control group before treatment [(11.21+/-1.11)%, (9.25+/-1.80) microg/L, (6.13+/-1.17) microg/L, all P<0.01]. After intervention, the levels of the above three indexes in both treatment groups were significantly decreased, and the decrease in DSM group [GMP140: (3.91+/- 0.57) microg/L , vWF: (3.86+/-0.71) microg/L, CD62P/CD61: (3.69+/-0.62)%] was more prominent than the CT group [GMP140: (2.30+/-0.33) microg/L, vWF: (2.72+/- 0.45) microg/L , CD62P/CD61: (2.24+/-0.45)%, all P<0.01], but they were higher than normal levels. CONCLUSION: DSM has the effect of inhibiting the activation of vascular endothelial cells and platelet. The medicine may be used to prevent thrombotic diseases.

[Effect of ulinastain on the expression of hemeoxygenase-1 in oleic acid-induced acute lung injury in rats].

OBJECTIVE: To explore the effect of ulinastain on the expression of hemeoxy genase-1 (HO-1) in oil acid-induced acute lung injury in rats. METHODS: The animal model of acute lung injury was established by oil acid. Thirty SD rats were randomly divided into 3 groups: the blank control group (A), the acute lung injury group (B) and the acute lung injury group (C) followed by injecting 100 mL/kg ulinastatin. Each group consisted of 10 rats. Group A were given 0.2 mL/kg natural solution through the trial vein; Group B and C were given 0.2 mL/kg oil-acid through trial vein, while group C were injected 100mL/kg ulinastatin by the peritoneal cavity after injecting oil acid. After 4 hours, the rates of respiration were counted and blood samples were cramped out through the heart puncture for blood gas analysis. The expressions of hemeoxygenase-1 and the pathologic construction changes were determined by HE staining in the lower right lung of rats in the 3 groups. RESULTS: The respiration dysfunction caused by oil acid could be prominently improved by ulinastain. There was only a little expression of hemeoxygenase-1 in the lung of Group A, but the expression increased in Group B and significatively increased in Group C. CONCLUSION: Ulinastatin may protect the rats from acute lung injury through increasing the expression of HO-1.