Bone-Differentiation-Associated Circ-Spen Regulates Death of Mouse Bone Marrow Mesenchymal Stem Cells by Inhibiting Apoptosis and Promoting Autophagy.

The role of estrogen receptor ß (ERß) in bone health is closely associated with its function in vivo, and ERß-/- mice have been widely utilized to explore the related influences. In this study, ERß-/- female mice were established to investigate the differential expression of circular RNAs (circRNAs) by RNA-Sequencing (RNA-Seq). Among these circRNAs, mmu_circ_0011379 (named Circ-Spen) exhibited high expression in ERß-/- female mice. However, the precise mechanism by which Circ-Spen regulates bone health remained unclear. This study identified Circ-Spen as a positive regulator of mouse bone marrow mesenchymal stem cell (mBMSC) viability. The expression of Circ-Spen was markedly increased in ERß-/- mice femurs tested by RT-qPCR. Moreover, Circ-Spen exhibited an enhanced expression during the bone formation process of mBMSCs. Qualitative experiments also demonstrated that Circ-Spen possessed a circular structure and was localized within the nucleus of mBMSCs. Functionally, it inhibited apoptosis via caspase-3, BCL-2, and BAX, while also promoting autophagy through BECN1 and P62 in mBMSCs tested by MTT assays, transmission electron microscopy (TEM), and Western blotting. These findings reveal the potential of targeting Circ-Spen as a promising therapeutic strategy for rejuvenating senescent mBMSCs and enhancing the efficiency of mBMSC transplantation, which lays the foundation for advancements in the field of bone therapy.

Single-cell transcriptomics uncover hub genes and cell-cell crosstalk in patients with hypertensive nephropathy.

Hypertensive nephropathy (HTN) is one of the leading causes of end-stage renal disease, yet the molecular mechanisms are still unknown. To explore novel mechanisms and gene targets for HTN, the gene expression profiles of renal biopsy samples obtained from 2 healthy living donor controls and 5 HTN patients were determined by single-cell RNA sequencing. Key hub genes expression were validated by the Nephroseq v5 platform. The HTN endothelium upregulated cellular adhesion genes (ICAM2 and CEACAM1), inflammatory genes (ETS2 and IFI6) and apoptosis related genes (CNN3). Proximal tubules in HTN highly expressed hub genes including BBOX1, TPM1, TMSB10, SDC4, and NUP58, which might be potential novel targets for proximal tubular injury. The upregulated genes in tubules of HTN were mainly participating in inflammatory signatures including IFN-γ signature, NF-κB signaling, IL-12 signaling and Wnt signaling pathway. Receptor-ligand interaction analysis indicated potential cell-cell crosstalk between endothelial cells or mesangial cells with other renal resident cells in HTN. Together, our data identify a distinct cell-specific gene expression profile, pathogenic inflammatory signaling and potential cell-cell communications between endothelial cells or mesangial cells with other renal resident cells in HTN. These findings may provide a promising novel landscape for mechanisms and treatment of human HTN.

Single-cell RNA sequencing reveals the transcriptomic landscape of kidneys in patients with ischemic acute kidney injury.

BACKGROUND: Ischemic acute kidney injury (AKI) is a common syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathogenesis of ischemic AKI remains incompletely understood, and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking. Here, this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing (scRNA-seq) analysis in kidneys. METHODS: In this study, scRNA-seq technology was applied to kidneys from two ischemic AKI patients, and three human public scRNA-seq datasets were collected as controls. Differentially expressed genes (DEGs) and cell clusters of kidneys were determined. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, as well as the ligand-receptor interaction between cells, were performed. We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion (I/R) injury induced AKI mice through immunohistochemistry staining. RESULTS: 15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control. The injured proximal tubules (PT) displayed a proapoptotic and proinflammatory phenotype. PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47 , RASSF4 , EBAG9 , IER3 , SASH1 , SEPTIN7 , and NUB1 , which have not been reported in ischemic AKI previously. Several hub genes were validated in kidneys from human AKI and renal I/R injury mice, respectively. Furthermore, PT highly expressed DEGs enriched in endoplasmic reticulum stress, autophagy, and retinoic acid-inducible gene I (RIG-I) signaling. DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain (NOD)-like receptor signaling, estrogen signaling, interleukin (IL)-12 signaling, and IL-17 signaling. Overexpressed genes in kidney-resident immune cells including macrophages, natural killer T (NKT) cells, monocytes, and dendritic cells were associated with leukocyte activation, chemotaxis, cell adhesion, and complement activation. In addition, the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI. CONCLUSION: Together, this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients, altered signaling pathways, and potential cell-cell crosstalk in the development of AKI. These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.

Clinical features and prognosis of MPO-ANCA and anti-GBM double-seropositive patients.

Background: Several lines of evidence implicate that there are distinct differences between patients with myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody (ANCA) and anti-glomerular basement membrane (GBM) antibody double-seropositive patients (DPPs) and single-positive patients. Hence, we conducted a retrospective study from a single center in China to analyze the clinical and pathological features, and prognosis of DPPs. Methods: 109 patients with MPO-ANCA-associated vasculitis (MPO-AAV), 20 DPPs and 23 patients diagnosed with anti-GBM disease from a large center in China were included in this study. The ratio of patients with renal biopsy in three groups were 100%, 50% and 100%, respectively. Their clinical and pathological characteristics, and outcomes were analyzed. The intensity of immune deposits in the kidney at diagnosis was detected by immunofluorescence (IF). Furthermore, multivariate Cox hazard model analysis was used to assess the clinical and histological predictors of end-stage renal disease (ESRD) and death for DPPs. Results: In our study, we found that patients in the DPPs group were older than the other two groups (p = 0.007, MPO-AAV vs. DPPs; p < 0.001, DPPs vs. anti-GBM). The DPPs group had a higher value of serum creatinine (p = 0.041) and lower estimated glomerular filtration rate (eGFR) (p = 0.032) compared with MPO-AAV patients. On the contrary, the DPPs group had a lower serum creatinine (p = 0.003) compared with patients with anti-GBM group. The proportion of patients with cardiac system involvement in the DPPs group was higher than anti-GBM patients (p = 0.014). Cellular crescents could be generally observed in renal biopsy of DPPs and patients with anti-GBM glomerulonephritis. In addition, Bowman's capsule rupture was more common in DPPs than MPO-AAV patients (p = 0.001). MPO-AAV had a better renal and overall survival outcome than DPPs (p < 0.001). There was no significant difference of renal and overall survival outcome between DPPs and patients with anti-GBM disease. The incidence of ESRD in DPPs was negatively associated with lymphocyte count (HR 0.153, 95% CI 0.027 to 0.872, p = 0.034) and eGFR (HR 0.847, 95% CI 0.726 to 0.989, p = 0.036). Elevated serum creatinine was confirmed as a risk factor of both renal (HR 1.003, 95% CI 1.000 to 1.005, p = 0.019) and patient survival in DPPs (HR1.461, 95% CI 1.050 to 2.033, p = 0.024). Conclusion: In summary, compared with anti-GBM disease, DPPs tended to involve multi-organ damage rather than limited to the kidney. It is highlighted that serologic DPPs have a worse renal and patient prognosis than MPO-AAV. Moreover, we found that the risk factors of renal survival of DPPs include low lymphocyte count, elevated serum creatinine and reduced eGFR, and serum creatinine can predict patient survival.

Preparation, Characterization, and In Vitro Release of Curcumin-Loaded IRMOF-10 Nanoparticles and Investigation of Their Pro-Apoptotic Effects on Human Hepatoma HepG2 Cells.

Curcumin (CUR) has a bright future in the treatment of cancer as a natural active ingredient with great potential. However, curcumin has a low solubility, which limits its clinical application. In this study, IRMOF-10 was created by the direct addition of triethylamine, CUR was loaded into IRMOF-10 using the solvent adsorption method, and the two were characterized using a scanning electron microscope (SEM), X-ray diffraction (XRD), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG) methods, and Brunauer-Emmett-Teller (BET) analysis. We also used the MTT method, 4',6-diamidino-2-phenylindole (DAPI) staining, the annexin V/PI method, cellular uptake, reactive oxygen species (ROS), and the mitochondrial membrane potential (MMP) to perform a safety analysis and anticancer activity study of IRMOF-10 and CUR@IRMOF-10 on HepG2 cells. Our results showed that CUR@IRMOF-10 had a CUR load of 63.96%, with an obvious slow-release phenomenon. The CUR levels released under different conditions at 60 h were 33.58% (pH 7.4) and 31.86% (pH 5.5). Cell experiments proved that IRMOF-10 was biologically safe and could promote curcumin entering the nucleus, causing a series of reactions, such as an increase in reactive oxygen species and a decrease in the mitochondrial membrane potential, thereby leading to cell apoptosis. In summary, IRMOF-10 is an excellent drug carrier and CUR@IRMOF-10 is an effective anti-liver cancer sustained-release preparation.

Kanglexin, a new anthraquinone compound, attenuates lipid accumulation by activating the AMPK/SREBP-2/PCSK9/LDLR signalling pathway.

Hyperlipidaemia is one of the major risk factors for atherosclerosis, coronary heart disease, stroke and diabetes. In the present study, we synthesized a new anthraquinone compound, 1,8-dihydroxy-3-succinic acid monoethyl ester-6-methylanthraquinone, and named it Kanglexin (KLX). The aim of this study was to evaluate whether KLX has a lipid-lowering effect and to explore the potential molecular mechanism. In this study, Sprague-Dawley rats were fed a high fat diet (HFD) for 5 weeks to establish a hyperlipidaemia model; then, the rats were orally administered KLX (20, 40, and 80 mg kg-1·d-1) or atorvastatin calcium (AT, 10 mg kg-1·d-1) once a day for 2 weeks. KLX had prominent effects on reducing blood lipids, hepatic lipid accumulation, body weight and the ratio of liver weight/body weight. Furthermore, KLXdramatically reduced the total cholesterol (TC) and triglyceride (TG) levels and lipid accumulation in a HepG2 cell model of dyslipidaemia induced by 1 mmol/L oleic acid (OA). KLX may decrease lipid levels by phosphorylating adenosine monophosphate-activated protein kinase (AMPK) and the downstream sterol regulatory element binding protein 2 (SREBP-2)/proprotein convertase subtilisin/kexin type 9 (PCSK9)/low-density lipoprotein receptor (LDLR) signalling pathway in the HFD rats and OA-treated HepG2 cells. The effects of KLX on the AMPK/SREBP-2/PCSK9/LDLR signalling pathway were abolished when AMPK was inhibited by compound C (a specific AMPK inhibitor) in HepG2 cells. In summary, KLX has an efficient lipid-lowering effect mediated by activation of the AMPK/SREBP-2/PCSK9/LDLR signalling pathway. Our findings may provide new insight into and evidence for the discovery of a new lipid-lowering drug for the prevention and treatment of hyperlipidaemia, fatty liver, and cardiovascular disease in the clinic.

Daming capsule, a hypolipidaemic drug, lowers blood lipids by activating the AMPK signalling pathway.

BACKGROUND/AIMS: Hyperlipidaemia is a major risk factor for cardiovascular and cerebrovascular diseases. Daming capsule (DMC), a medicine for lowering blood lipids, is marketed in China; however, its mechanism is unclear. The present study aimed to investigate the mechanism by which DMC reduces blood lipids. METHODS AND RESULTS: A rat model of hyperlipidaemia was established by feeding rats a high-fat diet (HFD), and the serum lipid levels were detected with an automatic biochemical analyser. DMC (162 mg/kg) and atorvastatin calcium (10 mg/kg) were orally administered to the hyperlipidaemic rats for 4 weeks. HFD feeding markedly induced increases in the levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-c); however, DMC treatment significantly decreased the levels of TC, TG, and LDL-c in rats serum. Meanwhile, the hepatic TC and TG levels, liver weight/body weight ratio, and body weight were significantly lower in the DMC-treated rats than in the HFD rats. Moreover, DMC significantly alleviated hepatomegaly, hepatic lipid deposition, and hepatic steatosis. The protein expression level of phospho-adenosine monophosphate-activated protein kinase (p-AMPK) (Thr172) in HFD rat livers was lower than that in normal rat livers, whereas it increased in the liver of the DMC-treated rats; however, the protein expression level of total-AMPK in the liver was not different among the groups. The AMPK-activating effect of DMC was blocked by Compound C (a specific AMPK inhibitor) in HepG2 cells. Additionally, DMC considerably increased peroxisome proliferator-activated receptor-alpha (PPARα) protein expression and lipoprotein lipase (LPL) transcription and concentration in the liver. This effect of DMC was also inhibited by Compound C in HepG2 cells. DMC also promoted LDL receptor (LDLR) protein expression by activating AMPK. We further found that DMC reduced the levels of TC and TG in oleic acid-treated HepG2 cells, and it restored the expression levels of p-AMPK, PPARα, LPL, and LDLR compared to the decreased levels observed in oleic acid-treated HepG2 cells. CONCLUSION: DMC lowered lipids in serum and the liver by activating AMPK. On the one hand, the activation of AMPK enhanced PPARα expression and LPL transcription to lead to the hydrolysis of TG; on the other hand, it increased LDLR protein expression to promote lipid metabolism.

Inflammatory myofibroblastic tumor of the larynx: report of a case and review of the literature.

Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm, most commonly seen in children and adolescents. It can occur in nearly every part of the body. Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm mostly seen in the lungs, but also in extrapulmonary sites. But it may rarely be seen in the vical cord. We report a case of a 73-year-old men presented with hoarseness and cough. Laryngoscopy reveals a large non-ulcerated, red subepithelial mass arising from the right vical cord. Magnetic resonance imaging (MRI) scan revealed a mass in the right vical cord, and magnetic resonance imaging (MRI) enhanced scan showed the mass of the right vical cord inhomogeneous enhancement. The patient underwent right cordectomy with KTP laser, and further assessment of the tissue demonstrated a pathologic diagnosis of IMT.

[The effect of envelope protein mutation on hepatitis B virus assemble].

OBJECTIVE: To investigate the effect of envelope protein mutation on HBV assembly. METHODS: The envelope protein mutated vectors were constructed by the molecular clone in vitro, and then transfected transiently in the cell HepG2. The expression and secretion of S protein was assay by ELISA. HBV DNA was quantitatively evaluated by PCR. After co-transfection with pHBV-mS1S and adwR9 the DNA was quantitatively evaluated by PCR. RESULTS: There was no significant difference in expression and secretion of S protein assayed by ELISA in the cytoplasm and supernatant among pHBV-mS1, pHBV-mS, pHBV-mS1S and the wild HBV adwR9 plasmid. The DNA detected by real-time fluorescence quantitative PCR from those the cytoplasm of mutants was higher than that from the wild HBV adwR9 cytoplasm, especially from the cytoplasm of pHBV-mS1S plasmid. However, the DNA detected by real-time fluorescence quantitative PCR from the supernatant of those mutants was lower than that from the wild HBV adwR9 supernatant, especially from pHBV-mS1S. The DNA detected by real-time fluorescence quantitative PCR from the supernatant co-transfected with pHBV-mS1S and adwR9 was lower than that from the supernatant co-transfected with pcDNA3 and adwR9. CONCLUSION: There was no effect of envelope protein mutation on the expression and secretion of S protein. Envelope protein mutation could interfere the assembly of HBV particle and cause reduction of secretion of HBV.

[Study on anti-HBV effects of genetically engineered replication-defective hepatitis B virus expressing dominant negative mutants of core protein].

BACKGROUND: To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein. METHODS: Two kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: The mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP. CONCLUSION: Dominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.

[Development of hygromycin-resistant packaging cell line for hepatitis B virus-derived vectors].

OBJECTIVE: To cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles. METHODS: Free of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: Hygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium. CONCLUSION: After the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.

[Replication and encapsidation of HBV mutants with the truncated C gene].

OBJECTIVE: To evaluate the replication and encapsidation of HBV mutants with the truncated C gene. METHODS: The HBV mutants with the truncated C gene were constructed by molecular cloning and PCR-based deletion in vitro. The replication and encapsidation of HBV mutants were investigated by Southern blotting, PCR and real-time fluorescence PCR respectively after transfecting the HBV mutants plasmid into HepG2 cells by using liposome. RESULTS: The C-truncated HBV mutant vectors were constructed successfully and confirmed exactly by clone sequencing and enzymes digestion. The C-truncated HBV mutants were replication defective, however, all types of HBV DNA could be detected positive in the cytoplasm and supernatant after co-transfecting the C-truncated HBV mutants plasmid and the helper constructs into HepG2 cells. The C-truncated HBV mutants were proved to produce 3-40 folds more progeny DNA than that of the wild-type HBV by DNA quantitative assay. CONCLUSION: The C-truncated HBV mutants are replication-deficient and could not replicate and encapsulate in the hepatocytes when transfected solely, however, the progeny HBV-variant viruses are encapsidated more effectively to secrete into supernatant when co-transfected with the helper construct which lacks part of 5 prime-proximal HBV RNA packaging signal Epsilon.

[The anti-HBV effect and mechanism of C gene truncated mutant in vitro].

OBJECTIVE: To explore the effect and mechanism on HBV replication in C gene truncated mutant. METHODS: Protein expression of C gene truncated vector and wild C gene vector were assay by SDS-PAGE Western blot. Constructed C gene truncated expression vector was cotransfected with wild HBV genome; virus load was detected by PCR in the culture medium and the cell. The formation of core particle was assay by Native western blot. RESULTS: The recombinant vectors can efficiently express. Virus load of the cotransfected group by pcDNA3-deltaC and adwR9 was lower than that of control group in the culture medium and the cell. Protein band of the co-expressed group by pcDNA3-deltaC and pcDNA3-C showed slightly weaker than that of the co-expressed group by pcDNA3 and pcDNA3-C. CONCLUSION: C gene truncated mutant could interfere with the formation of core particle and reduce of HBV replication

[Approach to transforming hepatitis B virus as a gene therapeutic vector].

OBJECTIVE: To evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy. METHODS: A fragment containing the small envelope gene of HBV was replaced with the reporter gene green fluorescent protein (GFP) to construct the recombinant HBV vector, which was transfected into HepG2 cells with liposome. The expression of GFP was observed with fluorescence microscope. The HBV cccDNA was testified using semi-nest PCR. The viral particles of the recombinant HBV in culture medium were detected by PCR as well as Southern blot. RESULTS: The HBV vector carrying the interesting gene of GFP could express the functional protein in the transfected hepatocytes. However, the recombinant HBV vector was replication-deficient, which could not be packed and replicated in the hepatocytes to secrete mature recombinant HBV particles carrying the interesting gene of GFP when transfected solely but could when cotransfected with the recombinant and helper construct which lacked part of 5'-proximal HBV RNA packaging signal epsilon. CONCLUSION: It is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.

[Construction and expression of recombinant retrovirus vector carrying HBV vector].

BACKGROUND: To explore the possibility of using retrovirus vector to carry HBV vector, and to prove that replication defective HBV could be normally packaged. METHODS: Two kinds of full length of mutant HBV gene, which express dominant negative mutants, were inserted into retrovirus vector. After recombinant retroviruses were harvested, they were used to infect Hep G2 and 2.2.15 cell line. Then the expression of HBV core antigen in the Hep G2 cell was examined by immune fluorescence, and the existence of recombinant HB virion in the culture medium was examined by PCR. RESULTS: High titer of recombinant retroviruses were obtained in the culture medium of transfected PA317 cell line. Core antigen was detectable in the recombinant retrovirus infected Hep G2 cell. Recombinant HB virion was detectable in the culture medium of recombinant retrovirus infected 2.2.15 cell. CONCLUSIONS: The results suggested that recombinant retrovirus could carry HBV vector and express HBV products. When structural protein is offered by wt-HBV, the recombinant retrovirus may function as HBV vector, therefore it could be used in anti?HBV gene therapy.