Tetrahydroxy stilbene glucoside rejuvenates aging hematopoietic stem cells with predilection for lymphoid differentiation via AMPK and Tet2.

INTRODUCTION: Aging of hematopoietic stem cells (HSCs) has emerged as an important challenge to human health. Recent advances have raised the prospect of rejuvenating aging HSCs via specific medical interventions, including pharmacological treatments. Nonetheless, efforts to develop such drugs are still in infancy until now. OBJECTIVES: We aimed to screen the prospective agents that can rejuvenate aging HSCs and explore the potential mechanisms. METHODS: We screened a set of natural anti-aging compounds through oral administration to sub-lethally irradiated mice, and identified 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG) as a potent rejuvenating agent for aging HSCs. Then naturally aged mice were used for the follow-up assessment to determine the HSC rejuvenating potential of TSG. Finally, based on the transcriptome and DNA methylation analysis, we validated the role of the AMP-activated protein kinase (AMPK)-ten-eleven-translocation 2 (Tet2) axis (the AMPK-Tet2 axis) as the underlying mechanisms of TSG for ameliorating HSCs aging. RESULTS: TSG treatment not only significantly increased the absolute number of common lymphoid progenitors (CLPs) along with B lymphocytes, but also boosted the HSCs/CLPs repopulation potential of aging mice. Further elaborated mechanism research demonstrated that TSG supplementation restored the stemness of aging HSCs, as well as promoted an epigenetic reprograming that was associated with an improved regenerative capacity and an increased rate of lymphopoiesis. Such effects were diminished when the mice were co-treated with an AMPK inhibitor, or when it was performed in Tet2 knockout mice as well as senescent cells assay. CONCLUSION: TSG is effective in rejuvenating aging HSCs by modulating the AMPK- Tet2 axis and thus represents a potential candidate for developing effective HSC rejuvenating therapies.

Ferritin-mediated mitochondrial iron homeostasis is essential for the survival of hematopoietic stem cells and leukemic stem cells.

Iron metabolism plays a crucial role in cell viability, but its relationship with adult stem cells and cancer stem cells is not fully understood. The ferritin complex, responsible for intracellular iron storage, is important in this process. We report that conditional deletion of ferritin heavy chain 1 (Fth1) in the hematopoietic system reduced the number and repopulation capacity of hematopoietic stem cells (HSCs). These effects were associated with a decrease in cellular iron level, leading to impaired mitochondrial function and the initiation of apoptosis. Iron supplementation, antioxidant, and apoptosis inhibitors reversed the reduced cell viability of Fth1-deleted hematopoietic stem and progenitor cells (HSPCs). Importantly, leukemic stem cells (LSCs) derived from MLL-AF9-induced acute myeloid leukemia (AML) mice exhibited reduced Fth1 expression, rendering them more susceptible to apoptosis induced by the iron chelation compared to normal HSPCs. Modulating FTH1 expression using mono-methyl fumarate increased LSCs resistance to iron chelator-induced apoptosis. Additionally, iron supplementation, antioxidant, and apoptosis inhibitors protected LSCs from iron chelator-induced cell death. Fth1 deletion also extended the survival of AML mice. These findings unveil a novel mechanism by which ferritin-mediated iron homeostasis regulates the survival of both HSCs and LSCs, suggesting potential therapeutic strategies for blood cancer with iron dysregulation.

A Versatile Chitosan-Based Hydrogel Accelerates Infected Wound Healing via Bacterial Elimination, Antioxidation, Immunoregulation, and Angiogenesis.

Drug-resistant bacterial infection of cutaneous wounds causes great harm to the human body. These infections are characterized by a microenvironment with recalcitrant bacterial infections, persistent oxidative stress, imbalance of immune regulation, and suboptimal angiogenesis. Treatment strategies available to date are incapable of handling the healing dynamics of infected wounds. A Schiff base and borate ester cross-linked hydrogel, based on phenylboronic acid-grafted chitosan (CS-PBA), dibenzaldehyde-grafted poly(ethylene glycol), and tannic acid (TA), is fabricated in the present study. Customized phenylboronic acid-modified zinc oxide nanoparticles (ZnO) are embedded in the hydrogel prior to gelation. The CPP@ZnO-P-TA hydrogel effectively eliminates methicillin-resistant Staphylococcus aureus (MRSA) due to the pH-responsive release of Zn2+ and TA. Killing is achieved via membrane damage, adenosine triphosphate reduction, leakage of intracellular components, and hydrolysis of bacterial o-nitrophenyl-ß-d-galactopyranoside. The CPP@ZnO-P-TA hydrogel is capable of scavenging reactive oxygen and nitrogen species, alleviating oxidative stress, and stimulating M2 polarization of macrophages. The released Zn2+ and TA also induce neovascularization via the PI3K/Akt pathway. The CPP@ZnO-P-TA hydrogel improves tissue regeneration in vivo by alleviating inflammatory responses, stimulating angiogenesis, and facilitating collagen deposition. These findings suggest that this versatile hydrogel possesses therapeutic potential for the treatment of MRSA-infected cutaneous wounds.

IiAGL6 participates in the regulation of stamen development and pollen formation in Isatis indigotica.

The AGL6 (AGMOUSE LIKE 6) gene is a member of the SEP subfamily and functions as an E-class floral homeotic gene in the development of floral organs. In this study, we cloned IiAGL6, the orthologous gene of AGL6 in Isatis indigotica. The constitutive expression of IiAGL6 in Arabidopsis thaliana resulted in a late-flowering phenotype and the development of curly leaves during the vegetative growth period. Abnormal changes in floral organ development were observed during the reproductive stage. In woad plants, suppression of IiAGL6 using TRV-VIGS (tobacco rattle virus-mediated virus-induced gene silencing) decreased the number of stamens and led to the formation of aberrant anthers. Similar changes in stamen development were also observed in miRNA-AGL6 transgenic Arabidopsis plants. Yeast two-hybrid and BiFC tests showed that IiAGL6 can interact with other MADS-box proteins in woad; thus, playing a key role in defining the identities of floral organs, particularly during stamen formation. These findings might provide novel insights and help investigate the biological roles of MADS transcription factors in I. indigotica.

Ultrasound Nanobubble Coupling Agent for Effective Noninvasive Deep-Layer Drug Delivery.

Conventional coupling agents (such as polyvinylpyrrolidone, methylcellulose, and polyurethane) are unable to efficiently transport drugs through the skin's dual barriers (the epidermal cuticle barrier and the basement membrane barrier between the epidermis and dermis) when exposed to ultrasound, hindering deep and noninvasive transdermal drug delivery. In this study, nanobubbles prepared by the double emulsification method and aminated hyaluronic acid are crosslinked with aldehyde-based hyaluronic acid by dynamic covalent bonding through the Schiff base reaction to produce an innovative ultrasound-nanobubble coupling agent. By amplifying the cavitation effect of ultrasound, drugs can be efficiently transferred through the double barrier of the skin and delivered to deep layers. In an in vitro model of isolated porcine skin, this agent achieves an effective penetration depth of 728 µm with the parameters of ultrasound set at 2 W, 650 kHz, and 50% duty cycle for 20 min. Consequently, drugs can be efficiently delivered to deeper layers noninvasively. In summary, this ultrasound nanobubble coupling agent efficiently achieves deep-layer drug delivery by amplifying the ultrasonic cavitation effect and penetrating the double barriers, heralding a new era for noninvasive drug delivery platforms and disease treatment.

Ultrasonic-controlled "explosive" hydrogels to precisely regulate spatiotemporal osteoimmune disturbance.

Spatiotemporal Immune disorder is a key factor leading to the failure of bone tissue healing. It is of vital importance to accurately suppress excessive peak immune response within 24-48 h of the injury and so regulate the spatiotemporal osteoimmune disturbance of bones. In this study, Ultrasound Controlled "Explosive" (UCE) hydrogels were prepared from gelatin-hyaluronic acid methacrylate hydrogels loaded with resveratrol nanobubbles produced by double emulsification through a condensation reaction. Such materials innovatively enable ultrasound-controlled RES release for precise regulation of spatiotemporal osteoimmune disorders. Under an ultrasonic power level of 1.5 W/cm2, the rate of effectively released RES through the blast of UCE hydrogels reached 38.14 %. And compared with the control group, the in vivo inhibition of inflammation and osteogenesis effects of UCE hydrogels were more effective, respectively. As suggested by the results, the excessive local inflammatory response was inhibited by the release of resveratrol, the temporospatial disorder of bone immune was precisely regulated, and as a result, the process of bone repair was accelerated. Altogether, this study confirms that the newly created UCE Hydrogels effectively promote bone repair by intervening peak inflammation during the early phase of fracture healing.

EVA1A regulates hematopoietic stem cell regeneration via ER-mitochondria mediated apoptosis.

Excessive protein synthesis upon enhanced cell proliferation frequently results in an increase of unfolded or misfolded proteins. During hematopoietic regeneration, to replenish the hematopoietic system, hematopoietic stem cells (HSCs) are activated and undergo a rapid proliferation. But how the activated HSCs respond to the proliferation pressure is still ambiguous; The proper control of the functional reservoir in the activated HSCs remains poorly understood. Here, we show a significant upregulation of EVA1A protein associated with the increase of ER stress during hematopoietic regeneration. Deletion of Eva1a significantly enhances the regeneration capacity of HSCs by inhibiting the ER stress-induced apoptosis. Mechanistically, the expression of EVA1A protein was upregulated by CHOP, and thereby promoted the ER-mitochondria interlinking via MCL1, which resulted in mitochondria-mediated apoptosis. These findings reveal a pathway for ER stress responses of HSCs by the EVA1A mediated apoptosis, which play an important role in HSCs regeneration.

Efficacy and safety of pyrotinib-containing regimen in the patients with HER2-positive metastatic breast cancer: A multicenter real-world study.

BACKGROUND: Pyrotinib, a novel irreversible epidermal growth factor receptor 2 (EGFR)/HER2 dual tyrosine kinase inhibitor, has shown promising antitumor efficacy with tolerable toxicity in HER2-positive metastatic breast cancer (MBC) in several clinical trials. However, the clinical trials do not usually well reflect the patients in real clinical settings. Despite several small-sample studies in real world, the data on pyrotinib as first-line and third-or-later-line treatment and the efficacy comparison of pyrotinib combined with different regimens are still lacking. Therefore, this study aimed to investigate the efficacy and safety of pyrotinib for the HER2-positive MBC in real world to replenish more comprehensive data. METHODS: A total of 172 HER2-positive MBC patients treated with pyrotinib-based therapy were recruited from multiple centers in nonclinical trial settings from September 2017 to June 2020. RESULTS: The median progression-free survival (mPFS) of 172 patients was 8.83 months. The patients, receiving first-line pyrotinib treatment, had the longest mPFS (20.93 months) compared with those receiving second-line (8.67 months, p = 0.0339) and third-or-later-line (7.13 months, p = 0.0075) treatments, respectively. Prior treatment with lapatinib (p = 0.012) and site of metastasis (visceral vs. nonvisceral) (p = 0.033) were the independent prognostic factors for PFS. The prior treatment with lapatinib compared with lapatinib-native treatment (5.96 vs. 10.97 months, p = 0.0036) and those with visceral metastasis compared with nonvisceral metastasis (8.40 vs. 23.70 months, p = 0.0138) had worse mPFS. Among 146 patients evaluated for efficacy, 2.1%, 58.9%, and 32.9% showed complete response, partial response, and stable disease, respectively. Adverse events occurred in 92.4% of the patients with 33.3% Grade 3 and higher adverse events and diarrhea (57.0%), anemia (44.8%), and leukopenia (40.7%) as the most frequent ones. CONCLUSIONS: Pyrotinib-containing regimen could effectively treat HER2-positive MBC with acceptable toxicity, including the patients who progressed after lapatinib treatment and with brain metastasis.

A retrospective comparative study of microwave ablation and sublobectomy in the treatment of early subpleural nonsmall cell lung cancer.

OBJECTIVE: Microwave ablation (MWA) is a safe and effective local therapy, however, its efficacy in stage I subpleural nonsmall cell lung cancer (NSCLC) compared to that of sublobar resection (SLR) is unclear. This study aimed to compare the efficacy of the two treatments for stage I NSCLC ≤1 cm from the pleura. METHODS: After propensity score matching (PSM), 70 patients with stage I subpleural NSCLC who underwent either SLR or MWA (35 patients each) from 2014 to 2018 were included. The margin pathology of SLR was negative. MWA reached a sufficient ablative margin. MWA group were stratified according to the minimal ablative margin, with 10 patients each in the 5-10 mm group and the >10 mm group after PSM. The local recurrence-free survival (LRFS), relapse-free survival (RFS), overall survival (OS), and treatment-related complications were compared. RESULTS: For patients with stage I subpleural NSCLC, the LRFS of patients in the SLR group (35.657 ± 0.338 months, 95% CI: 34.995-36.319) was significantly better than that in the MWA group (31.633 ± 1.574 months, 95% CI: 28.548-34.719, p = 0.021). The RFS was also significantly better in the SLR group (35.629 ± 0.338 months, 95% CI: 34.966-36.292) than in the MWA group (29.387 ± 1.866 months, 95% CI: 25.730-33.044, p = 0.007), but there were no significant differences in terms of the 3-year OS (p = 0.079) and incidence of complications (14.3% vs. 11.4%, p = 0.653). The minimal ablative margin of >10 mm was not significantly associated with the LRFS (p = 0.929). CONCLUSION: MWA for stage I subpleural NSCLC showed similar survival outcomes and complication rates to SLR, but poorer local tumor control.

Fabrication of gelatin-based and Zn2+-incorporated composite hydrogel for accelerated infected wound healing.

Gelatin-based hydrogels have a broad range of biomedical fields due to their biocompatibility, convenience for chemical modifications, and degradability. However, gelatin-based hydrogels present poor antibacterial ability that hinders their applications in treating infected wound healing. Herein, a series of multifunctional hydrogels (Gel@Zn) were fabricated through free-radical polymerization interaction based on gelatin methacrylate (GelMA) and dopamine methacrylate (DMA), and then immersed them into zinc nitrate solutions based on the metal coordination and ionic bonding interaction. These designed hydrogels wound dressings show strong antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) by increasing intracellular reactive oxygen species (ROS) level and changing bacterial membrane permeability. Meanwhile, the hydrogels exhibit good cytocompatibility, enhance the adhesion, proliferation, and migration of NIH-3T3 cells. Furthermore, Gel@Zn-0.08 (0.08 â€‹M Zn2+ immersed with Gel sample) presents a good balance between antibacterial effect, cell viability, and hemolytic property. Compared with 3 â€‹M commercial dressings, Gel@Zn-0.04, and Gel@Zn-0.16, the Gel@Zn-0.08 could significantly improve the healing process of S. aureus-infected full-thickness wounds via restrained the inflammatory responses, enhanced epidermis and granulation tissue information, and stimulated angiogenesis. Our study indicates that the Zn-incorporated hydrogels are promising bioactive materials as wound dressings for infected full-thickness wound healing and skin regeneration.

Low Expression of Long Noncoding RNA SLC26A4 Antisense RNA 1 Is an Independent Prognostic Biomarker and Correlate of Immune Infiltrates in Breast Cancer.

BACKGROUND Aberrant expression of long noncoding RNA (lncRNA) SLC26A4 antisense RNA 1 (SLC26A4-AS1) plays an important role in some cancer types. However, the clinical significance of SLC26A4-AS1 in patients with breast cancer (BC) and the possible regulatory mechanisms of SLC26A4-AS1 are unclear. MATERIAL AND METHODS Statistical analysis was used to assess the correlation between SLC26A4-AS1 expression and patients' clinical characteristics. The Kaplan-Meier method and Cox regression analysis were used to assess the correlation between SLC26A4-AS1 expression and prognosis. Gene set enrichment analysis (GSEA) and immuno-infiltration analysis were used to investigate the possible regulatory mechanisms of SLC26A4-AS1. RESULTS Low SLC26A4-AS1 expression in BC was associated with age (P<0.001), estrogen-receptor status (P<0.001), PAM50 (P<0.001), and menopause status (P<0.001). Low SLC26A4-AS1 expression predicted a poorer overall survival (OS) (hazard ratio [HR]: 0.56; 95% confidence interval [CI]: 0.40-0.78; P=0.001) and disease-specific survival (DSS) (HR: 0.57; 95% CI: 0.37-0.88; P=0.011). Also, SLC26A4-AS1 expression (HR: 0.298; 95% CI: 0.154-0.579; P<0.001) was independently correlated with OS in patients with BC. SLC26A4-AS1 was related to CYP2E1 reactions, protein export, mitochondrial_ciii_assembly, formation of adenosine triphosphate by chemiosmotic coupling, budding and maturation of HIV virion, cristae formation, biocarta proteasome pathway, endosomal sorting complex required for transport, and histone modification. SLC26A4-AS1 expression was associated with some types of immune infiltrating cells. CONCLUSIONS SLC26A4-AS1 expression was significantly associated with poor survival and immune infiltration in patients with BC. It may be a promising prognostic biomarker for BC.

LIPUS inhibits inflammation and catabolism through the NF-κB pathway in human degenerative nucleus pulposus cells.

BACKGROUND: Low-intensity pulsed ultrasound (LIPUS) is a safe and noninvasive rehabilitative physical therapy with anti-inflammatory effects. The current study investigated the effect of LIPUS on the inflammation of nucleus pulposus (NP) cells and its underlying mechanism. METHODS: Human NP cells were acquired from lumbar disc herniation tissue samples and cultured for experiments. Human NP cells were treated with LPS and then exposed to LIPUS (15 mW/cm2, 30 mW/cm2 and 60 mW/cm2) for 20 min daily for 3 days to determine the appropriate intensity to inhibit the expression of the inflammatory factors TNF-α and IL-1ß. The gene and protein expression of aggrecan, collagen II, MMP-3 and MMP-9 was measured by real-time PCR and western blotting, respectively. The activity of the nuclear factor-kappa B (NF-κB) pathway was examined by western blotting and immunofluorescence. After pretreatment with the NF-κB inhibitor PDTC, the expression of TNF-α, IL-1ß, MMP-3 and MMP-9 was measured by real-time PCR. RESULTS: LIPUS at intensities of 15 mW/cm2, 30 mW/cm2 and 60 mW/cm2 inhibited LPS-induced NP cell expression of the inflammatory factors TNF-α and IL-1ß, especially at 30 mW/cm2. LIPUS significantly upregulated the gene and protein expression of aggrecan and collagen II and downregulated the gene and protein expression of MMP-3 and MMP-9 in LPS-induced NP cells. The NF-κB signaling pathway was inhibited by LIPUS through inhibiting the protein expression of p-P65 and the translocation of P65 into the nucleus in LPS-induced NP cells. In addition, LIPUS had similar effects as the NF-κB inhibitor PDTC by inhibiting the NF-κB signaling pathway, inflammation and catabolism in LPS-induced human degenerative nucleus pulposus cells. CONCLUSION: LIPUS inhibited inflammation and catabolism through the NF-κB pathway in human degenerative nucleus pulposus cells.

Overexpression of miR-338-5p in exosomes derived from mesenchymal stromal cells provides neuroprotective effects by the Cnr1/Rap1/Akt pathway after spinal cord injury in rats.

Growing evidence has shown that microRNAs (miRNAs) play crucial roles in the physiopathology of spinal cord injury (SCI). Recent studies have confirmed that miR-338-5p regulates myelination, suggesting a potential role in the treatment of SCI. However, the molecular mechanism of miR-338-5p on SCI is still unknown. Recently, exosomes have emerged as an ideal vector to deliver therapeutic molecules such as miRNAs. Here, we explored the effects of miR-338-5p-overexpressing exosomes derived from bone marrow-derived mesenchymal stromal cells (BMSCs) on SCI. In vivo, a model of contusion SCI in rats was established, and we observed that overexpression of miR-338-5p in exosomes profoundly increased the expression levels of neurofilament protein-M and growth-associated protein-43 and decreased those of myelin-associated glycoprotein and glial fibrillary acidic protein, which provided neuroprotective effects after acute SCI. In an in vitro study, we found that overexpression of miR-338-5p in exosomes repressed cell apoptosis following H2O2-induced oxidative stress injury in PC12 cells. Additionally, we confirmed that cannabinoid receptor 1 (Cnr1) was the target gene of miR-338-5p by dual-luciferase reporter assays and that Rap1 was the downstream gene by the KEGG pathway analysis. We found that miR-338-5p increased cAMP accumulation as a consequence of downregulated expression of the target gene Cnr1, and then, Rap1 was activated by cAMP. Eventually, the activation of the PI3K/Akt pathway attenuated cell apoptosis and promoted neuronal survival by cAMP-mediated Rap1 activation. In brief, these findings showed that exosomes overexpressing miR-338-5p were a promising treatment strategy for SCI.

[Experimental study on the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits].

OBJECTIVE: To investigate the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits. METHODS: Twenty-six 3-month-old New Zealand rabbits, 2.0-2.5 kg in weight, were used to establish the model of intervertebral disc degeneration at L 3, 4, L 4, 5, and L 5, 6 by transabdominal needle puncture. At 4 weeks after operation, the 24 rabbits were randomly divided into 4 groups after successful modeling, which checked by MRI. The target intervertebral discs of each group were injected with zinc finger protein A20 overexpressed adenovirus (Ov-A20 group), empty carrier adenovirus (NC group), phosphate buffer saline (control group), and shRNA-A20 adenovirus (Sh-A20 group). The biological responses of animals in each group were comprehensive scored before 1 day of injection and after 1, 2, 3, and 6 days of injection. At 2, 4, and 8 weeks after injection, the animals in each group were observed by MRI to obtain the exact T2 relaxation time (T2 signal value). After MRI examination, the animals were killed to take the degenerative intervertebral disc tissue; and the tissue was detected by Alcian blue staining to observed the intervertebral disc degeneration. The expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were detected by immunohistochemistry staining. The expressions of zinc finger protein A20, nuclear factor κB binding protein [P65, phosphate P65 (P-P65), collagen Ⅱ, aggrecan], inflammatory factors [tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß)], autophagy-related protein [LC3 (LC3Ⅱ/LC3Ⅰ) and P62] were detected by Western blot. RESULTS: The comprehensive score of biological response in each group after injection was significantly lower than that before injection ( P<0.05). At 6 days after injection, the comprehensive score of biological response in the Sh-A20 group was significantly lower than that in other groups ( P<0.05), and there was no significant difference among other groups ( P>0.05). The detection of MRI showed that the T2 signal value in the Ov-A20 group was the highest at 2, 4, and 8 weeks after injection ( P<0.05), and the T2 signal value in the Sh-A20 group was the lowest at 2 and 4 weeks after injection ( P<0.05). There was no significant difference between other groups ( P>0.05). Alcian blue staining showed that the expression of aggrecan was the highest in Ov-A20 group and the lowest in Sh-A20 group at 4 weeks ( P<0.05); the expression of aggrecan in Ov-A20 group was the highest at 8 weeks ( P<0.05), and there was no significant difference between other groups ( P>0.05). Immunohistochemical staining showed that the expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were the highest in Ov-A20 group and lowest in Sh-A20 group ( P<0.05). Western blot showed that the expressions of zinc finger protein A20, collagen Ⅱ, aggrecan, and LC3 (LC3Ⅱ/LC3Ⅰ) proteins were the highest in the Ov-A20 group and the lowest in Sh-A20 group ( P<0.05), while the expressions of P-P65, TNF-α, IL-1ß, and P62 proteins were the lowest in Ov-A20 group and the highest in Sh-A20 group ( P<0.05). There was no significant difference in the expression of p65 protein between groups ( P>0.05). CONCLUSION: Zinc finger protein A20 can effectively regulate the process of lumbar intervertebral disc degeneration in rabbits by inhibiting inflammation.

Protein S-nitrosylation regulates proteostasis and viability of hematopoietic stem cell during regeneration.

Hematopoietic stem cells (HSCs) regenerate blood cells upon hematopoietic injuries. During homeostasis, HSCs are maintained in a low reactive oxygen species (ROS) state to prevent exhaustion. However, the role of nitric oxide (NO) in controlling HSC regeneration is still unclear. Here, we find increased NO during HSC regeneration with an accumulation of protein aggregation. S-nitrosoglutathione reductase (GSNOR)-deleted HSCs exhibit a reduced reconstitution capacity and loss of self-renewal after chemotherapeutic injury, which is resolved by inhibition of NO synthesis. Deletion of GSNOR enhances protein S-nitrosylation, resulting in an accumulation of protein aggregation and activation of unfolded protein response (UPR). Treatment of taurocholic acid (TCA), a chemical chaperone, rescues the regeneration defect of Gsnor-/- HSCs after 5-fluorouracil (5-FU) treatment. Deletion of C/EBP homologous protein (Chop) restores the reconstitution capacity of Gsnor-/- HSCs. These findings establish a link between S-nitrosylation and protein aggregation in HSC in the context of blood regeneration.

A20 regulates inflammation through autophagy mediated by NF-κB pathway in human nucleus pulposus cells and ameliorates disc degeneration in vivo.

Intervertebral disc degeneration (IDD) is closely related to loss of the extracellular matrix (ECM), apoptosis and inflammation in nucleus pulposus cells (NPCs). It has been reported that Zinc finger protein A20/TNFAIP3 (A20) can inhibit the activity of the NF-κB pathway and promote autophagy. Therefore, we speculated that A20 can regulate inflammation and ameliorate IDD through autophagy mediated by NF-κB in human NPCs. Our results indicated that the expression of A20 and inflammatory factors in IDD tissues was increased. A20 is an essential negative regulator in the NF-κB pathway. Constructed adenoviral shRNA and overexpression vectors for A20 could effectively regulate the inflammation, autophagy, and activity of NF-κB, which in turn affected the progression of IDD. Inhibition of NF-κB on the basis of knocking down A20 results in increased autophagy, suggesting that A20-regulated autophagy was mediated by NF-κB. In vivo, A20 overexpression could ameliorate the progression of IDD and promote autophagy at the same time, while deletion of A20 leads to low levels of autophagy and severe degeneration. In summary, A20 plays an important role in inhibiting inflammation through autophagy mediated by NF-κB in NPCs and ameliorating IDD.

Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism.

BACKGROUND: Small vessel vasculitis (SVV) is a group of systemic autoimmune diseases that are mediated by neutrophil extracellular traps (NETs) in response to cathelicidin LL37, an aging molecular marker, which could be induced by telomere dysfunction. Therefore, in this study, we evaluated the hypothesis that telomere dysfunction in neutrophils may promote SVV via an LL37-NETs-dependent mechanism. METHODS: We contrasted the release of neutrophil NETs from mice with telomere dysfunction, mice with DNA damage and wide-type mice. Neutrophil telomere length, the expression of LL37, and the formation of NETs were measured in SVV patients and healthy controls (HCs). The co-expression of γH2AX, LL37, and NETs were detected in SVV patients to evaluate the association of the immune aging of neutrophils and pro-inflammatory conditions. LL37 inhibitor was used to verify its key role in NETs release in SVV patients and DNA damage mice. RESULTS: We found that NETs were over-induced by telomere dysfunction and DNA damage in mice, which may be associated with a marked increase in LL37. For patients with SVV, telomeres in neutrophils were significantly shortened, which was also associated with higher levels of LL37 and NETs. Inhibition of LL37 reduced the NETs released from neutrophils. CONCLUSIONS: Taken together, the results of these studies suggest that dysfunction of telomeres may promote SVV through the mechanism of LL37-dependent NETs. Thus, targeting the LL37-NETs may be a novel therapy for SVV.

Protective effects of autophagy and NFE2L2 on reactive oxygen species-induced pyroptosis of human nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is characterized by the decrease of nucleus pulposus cells (NPCs). With the increase of the degree of degeneration, the reactive oxygen species (ROS) in nucleus pulposus tissue increases. Pyroptosis is a newly discovered form of cell death and its relationship with oxidative stress in NPCs remains unclear. This study was performed to investigate the mechanisms of pyroptosis of NPCs under oxidative stress. NPCs were isolated from IDD patients by surgical treatment. Pyroptosis related proteins like NLR family pyrin domain containing 3(NLRP3) and PYD and CARD domain containing (PYCARD) were detected by western blot, and membrane pore formation was observed by hochest33342/PI double staining or scanning electron microscope. The results showed that ROS induced the pyroptosis of NPCs and it depended on the expression of NLRP3 and PYCARD. The increased ROS level also increased transcription factor nuclear factor, erythroid 2 like 2 (NFE2L2, Nrf2) and the autophagy of NPCs, both of which attenuated the pyroptosis. In summary, ROS induces the pyroptosis of NPCs through the NLRP3/ PYCARD pathway, and establishes negative regulation by increasing autophagy and NFE2L2. These findings may provide a better understanding of the mechanism of IDD and potential therapeutic approaches for IDD treatment.

HO-1 overexpression alleviates senescence by inducing autophagy via the mitochondrial route in human nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.

The transcription factor Zfp281 sustains CD4+ T lymphocyte activation through directly repressing Ctla-4 transcription.

The expression of coinhibitory receptors, such as CTLA-4, on effector T cells is a key mechanism for the negative regulation of T-cell activation. However, the transcriptional regulation of CTLA-4 is not well understood. Zfp281, a C2H2 zinc finger protein, is a negative regulator of pluripotency maintenance of embryonic stem cells. Nevertheless, the function of Zfp281 in differentiated cells has not been studied. We generated Zfp281 conditional knockout mice in which the function of the Zfp281 gene was conditionally disrupted by the Cd4Cre transgene to study its impact on T cell function. Zfp281 had no effect on T-cell development, but CD4+ T cell activation and cytokine production were impaired due to diminished T-cell receptor signaling. Furthermore, Zfp281 deficiency inhibited in vivo T cell responses to Listeria monocytogenes infection. Using genome-wide expression profiling assays, we determined that Zfp281 repressed Ctla-4 expression by directly binding to GC-rich sites in its promoter, which inhibited the negative feedback of T cell activation. In line with this result, CTLA-4 blockade and shRNA knockdown partly rescued the reduced cytokine production caused by Zfp281 deficiency. These findings indicate that Zfp281 sustains CD4+ T lymphocyte activation by directly repressing Ctla-4 transcription.