hP-MSCs attenuate severe acute pancreatitis in mice via inhibiting NLRP3 inflammasome-mediated acinar cell pyroptosis.

BACKGROUND: Severe acute pancreatitis (SAP) is a serious gastrointestinal disease that is facilitated by pancreatic acinar cell death. The protective role of human placental mesenchymal stem cells (hP-MSCs) in SAP has been demonstrated in our previous studies. However, the underlying mechanisms of this therapy remain unclear. Herein, we investigated the regularity of acinar cell pyroptosis during SAP and investigated whether the protective effect of hP-MSCs was associated with the inhibition of acinar cell pyroptosis. METHODS: A mouse model of SAP was established by the retrograde injection of sodium taurocholate (NaTC) solution in the pancreatic duct. For the hP-MSCs group, hP-MSCs were injected via the tail vein and were monitored in vivo. Transmission electron microscopy (TEM) was used to observe the pyroptosis-associated ultramorphology of acinar cells. Immunofluorescence and Western blotting were subsequently used to assess the localization and expression of pyroptosis-associated proteins in acinar cells. Systemic inflammation and local injury-associated parameters were evaluated. RESULTS: Acinar cell pyroptosis was observed during SAP, and the expression of pyroptosis-associated proteins initially increased, peaked at 24 h, and subsequently showed a decreasing trend. hP-MSCs effectively attenuated systemic inflammation and local injury in the SAP model mice. Importantly, hP-MSCs decreased the expression of pyroptosis-associated proteins and the activity of the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in acinar cells. CONCLUSIONS: Our study demonstrates the regularity and important role of acinar cell pyroptosis during SAP. hP-MSCs attenuate inflammation and inhibit acinar cell pyroptosis via suppressing NLRP3 inflammasome activation, thereby exerting a protective effect against SAP.

miR-134-5p inhibits osteoclastogenesis through a novel miR-134-5p/Itgb1/MAPK pathway.

Osteoporosis affects approximately 200 million people and severely affects quality of life, but the exact pathological mechanisms behind this disease remain unclear. Various miRNAs have been shown to play a predominant role in the regulation of osteoclast formation. In this study, we explored the role of miR-134-5p in osteoclastogenesis both in vivo and in vitro. We constructed an ovariectomized (OVX) mouse model and performed microarray analysis using bone tissue from OVX mice and their control counterparts. Quantitative RT-PCR data from bone tissue and bone marrow macrophages (BMMs) confirmed the decreased expression of miR-134-5p in OVX mice observed in microarray analysis. In addition, a decrease in miR-134-5p was also observed during induced osteoclastogenesis of BMMs collected from C57BL/6N mice. Through transfection with miR-134-5p agomirs and antagomirs, we found that miR-134-5p knockdown significantly accelerated osteoclast formation and cell proliferation and inhibited apoptosis. Furthermore, a luciferase reporter assay showed that miR-134-5p directly targets the integrin surface receptor gene Itgb1. Cotransfection with Itgb1 siRNA reversed the effect of the miR-134-5p antagomir in promoting osteoclastogenesis. Moreover, the abundance levels of MAPK pathway proteins phosphorylated-p38 (p-p38) and phosphorylated-ERK (p-ERK) were significantly increased after transfection with the miR-134-5p antagomir but decreased after transfection with the miR-134-5p agomir or Itgb1 siRNA, which indicated a potential relationship between the miR-134-5p/Itgb1 axis and the MAPK pathway. Collectively, these results revealed that miR-134-5p inhibits osteoclast differentiation of BMMs both in vivo and in vitro and that the miR-134-5p/Itgb1/MAPK pathway might be a potential target for osteoporosis therapy.

A retrospective study comparing DALK and PKP in the treatment of necrotizing stromal keratitis.

PURPOSE: To compare the clinical outcomes of deep anterior lamellar keratoplasty (DALK) and penetrating keratoplasty (PKP) in the treatment of necrotizing stromal keratitis (NSK). METHODS: A retrospective study of NSK patients who underwent keratoplasty between January 2015 and December 2017 in the Third Xiangya Hospital was carried out. Data including preoperative and postoperative best corrected visual acuity (BCVA), intraocular pressure, graft survival rates, corneal endothelial cell density, corneal topography and thickness were reviewed and analyzed by SPSS 23.0 software. RESULTS: Fifty patients were involved. Twenty-five patients received DALK, and the other half received PKP. The average follow-up period was 10.28 ± 5.92 months. At the end of the follow-up period, there were no significant differences in postoperative BCVA, recurrence of virus, graft rejection or graft failure between the two groups. There were also no significant differences in average central corneal thickness postoperatively at 3 months. However, the average corneal endothelial cell density at 3 months was significantly higher in the DALK group (2121.12 ± 450.80 cell/mm2 in the DALK group versus 1812.16 ± 340.38 cell/mm2 in the PKP group, P = 0.009). CONCLUSION: Both DALK and PKP could increase visual acuity and prevent the progression of NSK. There were no significant differences between DALK and PKP in postoperative BCVA, rate of rejection, graft failure or recurrence rate. DALK significantly reduced the loss of corneal endothelial cells.

All-trans retinoic acid stimulates the secretion of TGF-ß2 via the phospholipase C but not the adenylyl cyclase signaling pathway in retinal pigment epithelium cells.

BACKGROUND: By investigating that (i) all-trans retinoic acid (ATRA) affects human retinal pigment epithelium (RPE) in expressing and secreting transforming growth factor (TGF)-ß2 and (ii) U73122 (phospholipase C inhibitor) and SQ22536 (adenylyl cyclase inhibitor) regulate the ATRA-induced secretion of TGF-ß2 in human RPE, we sought to interpret the signaling pathway of ATRA in promoting the development of myopia. METHODS: The RPE cell line (D407) was treated with (i) ATRA (10 µM), (ii) U73122 (5-40 µM) and ATRA (10 µM), or (iii) SQ22536 (5-40 µM) and ATRA (10 µM). The control group was no-treated. After stimulated at 2, 4, 8, 16, 24, and 48 h, The expression and secretion of TGF-ß2 was detected. RESULTS: TGF-ß2 in the cytoplasm was time-dependent increased by ATRA (p < 0.001). A time-dependent increase in the TGF-ß2 protein of the supernatant was induced by ATRA (p < 0.001). U73122 (in the range of 5 to 40 µM) could suppress the secretion of TGF-ß2 induced by ATRA (p < 0.001), and 40 µM U73122 could completely inhibit the up-regulated effect of 10 µM ATRA. However, SQ22536 (in the range of 5 to 40 µM) had no impact on the secretion of TGF-ß2 induced by ATRA (p > 0.05). CONCLUSIONS: In RPE cells, ATRA stimulates the secretion of TGF-ß2 via the phospholipase C signaling pathway but not the adenylyl cyclase signaling pathway. U73122 may inhibit the promotion of ATRA in the development of myopia.

Deubiquitinase Mysm1 regulates neural stem cell proliferation and differentiation by controlling Id4 expression.

Neural stem cells (NSCs) are critical for brain development and maintenance of neurogenesis. However, the molecular mechanisms that regulate NSC proliferation and differentiation remain unclear. Mysm1 is a deubiquitinase and is essential for the self-renewal and differentiation of several stem cells. It is unknown whether Mysm1 plays an important role in NSCs. Here, we found that Mysm1 was expressed in NSCs and its expression was increased with age in mice. Mice with Mysm1 knockdown by crossing Mysm1 floxed mice with Nestin-Cre mice exhibited abnormal brain development with microcephaly. Mysm1 deletion promoted NSC proliferation and apoptosis, resulting in depletion of the stem cell pool. In addition, Mysm1-deficient NSCs skewed toward neurogenesis instead of astrogliogenesis. Mechanistic investigations with RNA sequencing and genome-wide CUT&Tag analysis revealed that Mysm1 epigenetically regulated Id4 transcription by regulating histone modification at the promoter region. After rescuing the expression of Id4, the hyperproliferation and imbalance differentiation of Mysm1-deficient NSCs was reversed. Additionally, knockdown Mysm1 in aged mice could promote NSC proliferation. Collectively, the present study identified a new factor Mysm1 which is essential for NSC homeostasis and Mysm1-Id4 axis may be an ideal target for proper NSC proliferation and differentiation.

GDNF promotes the proliferation and osteogenic differentiation of jaw bone marrow mesenchymal stem cells via the Nr4a1/PI3K/Akt pathway.

How to efficiently regenerate jawbone defects caused by trauma, jaw osteomyelitis, tumors, or intrinsic genetic diseases is still challenging. Ectoderm-derived jawbone defect has been reported to be regenerated by selectively recruiting cells from its embryonic origin. Therefore, it is important to explore the strategy for promoting ectoderm-derived jaw bone marrow mesenchymal stem cells (JBMMSCs) on the repair of homoblastic jaw bone. Glial cell-derived neurotrophic factor (GDNF) is an important growth factor and is essential in the process of proliferation, migration and differentiation of nerve cells. However, whether GDNF promoting the function of JBMMSCs and the relative mechanism are not clear. Our results showed that activated astrocytes and GDNF were induced in the hippocampus after mandibular jaw defect. In addition, the expression of GDNF in the bone tissue around the injured area was also significantly increased after injury. Data from in vitro experiments demonstrated that GDNF could effectively promote the proliferation and osteogenic differentiation of JBMMSCs. Furthermore, when implanted in the defected jaw bone, JBMMSCs pretreated with GDNF exhibited enhanced repair effect compared with JBMMSCs without treatment. Mechanical studies found that GDNF induced the expression of Nr4a1 in JBMMSCs, activated PI3K/Akt signaling pathway and then enhanced the proliferation and osteogenic differentiation capacities of JBMMSCs. Our studies reveal that JBMMSCs are good candidates for repairing jawbone injury and pretreated with GDNF is an efficient strategy for enhancing bone regeneration.

Ectoderm-derived frontal bone mesenchymal stem cells promote traumatic brain injury recovery by alleviating neuroinflammation and glutamate excitotoxicity partially via FGF1.

BACKGROUND: Traumatic brain injury (TBI) leads to cell and tissue impairment, as well as functional deficits. Stem cells promote structural and functional recovery and thus are considered as a promising therapy for various nerve injuries. Here, we aimed to investigate the role of ectoderm-derived frontal bone mesenchymal stem cells (FbMSCs) in promoting cerebral repair and functional recovery in a murine TBI model. METHODS: A murine TBI model was established by injuring C57BL/6 N mice with moderate-controlled cortical impact to evaluate the extent of brain damage and behavioral deficits. Ectoderm-derived FbMSCs were isolated from the frontal bone and their characteristics were assessed using multiple differentiation assays, flow cytometry and microarray analysis. Brain repairment and functional recovery were analyzed at different days post-injury with or without FbMSC application. Behavioral tests were performed to assess learning and memory improvements. RNA sequencing analysis, immunofluorescence staining, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to examine inflammation reaction and neural regeneration. In vitro co-culture analysis and quantification of glutamate transportation were carried out to explore the possible mechanism of neurogenesis and functional recovery promoted by FbMSCs. RESULTS: Ectoderm-derived FbMSCs showed fibroblast like morphology and osteogenic differentiation capacity. FbMSCs were CD105, CD29 positive and CD45, CD31 negative. Different from mesoderm-derived MSCs, FbMSCs expressed the ectoderm-specific transcription factor Tfap2ß. TBI mice showed impaired learning and memory deficits. Microglia and astrocyte activation, as well as neural damage, were significantly increased post-injury. FbMSC application ameliorated the behavioral deficits of TBI mice and promoted neural regeneration. RNA sequencing analysis showed that signal pathways related to inflammation decreased, whereas those related to neural activation increased. Immunofluorescence staining and qRT-PCR data revealed that microglial activation and astrocyte polarization to the A1 phenotype were suppressed by FbMSC application. In addition, FGF1 secreted from FbMSCs enhanced glutamate transportation by astrocytes and alleviated the cytotoxic effect of excessive glutamate on neurons. CONCLUSIONS: Ectoderm-derived FbMSC application significantly alleviated neuroinflammation, brain injury, and excitatory toxicity to neurons, improved cognition and behavioral deficits in TBI mice. Therefore, ectoderm-derived FbMSCs could be ideal therapeutic candidates for TBI which mostly affect cells from the same embryonic origins as FbMSCs.

Genetic and Pharmacological Inhibition of Astrocytic Mysm1 Alleviates Depressive-Like Disorders by Promoting ATP Production.

Major depressive disorder (MDD) is a leading cause of disability worldwide. A comprehensive understanding of the molecular mechanisms of this disorder is critical for the therapy of MDD. In this study, it is observed that deubiquitinase Mysm1 is induced in the brain tissues from patients with major depression and from mice with depressive behaviors. The genetic silencing of astrocytic Mysm1 induced an antidepressant-like effect and alleviated the osteoporosis of depressive mice. Furthermore, it is found that Mysm1 knockdown led to increased ATP production and the activation of p53 and AMP-activated protein kinase (AMPK). Pifithrin α (PFT α) and Compound C, antagonists of p53 and AMPK, respectively, repressed ATP production and reversed the antidepressant effect of Mysm1 knockdown. Moreover, the pharmacological inhibition of astrocytic Mysm1 by aspirin relieved depressive-like behaviors in mice. The study reveals, for the first time, the important function of Mysm1 in the brain, highlighting astrocytic Mysm1 as a potential risk factor for depression and as a valuable target for drug discovery to treat depression.