An Approach to Incorporate Multi-Enzyme Digestion into C-TAILS for C-Terminomics Studies.

Protein C-termini study is still a challenging task and far behind its counterpart, N-termini study. MS based C-terminomics study is often hampered by the low ionization efficiency of C-terminal peptides and the lack of efficient enrichment methods. We previously optimized the C-terminal amine-based isotope labeling of substrates (C-TAILS) method and identified 369 genuine protein C-termini in Escherichia coli. A key limitation of C-TAILS is that the prior protection of amines and carboxylic groups at protein level makes Arg-C as the only specific enzyme in practice. Herein, we report an approach combining multi-enzyme digestion and C-TAILS, which significantly increases the identification rate of C-terminal peptides and consequently improves the applicability of C-TAILS in biological studies. We carry out a systematic study and confirm that the omission of the prior amine protection at protein level has a negligible influence and allows the application of multi-enzyme digestion. We successfully apply five different enzyme digestions to C-TAILS, including trypsin, Arg-C, Lys-C, Lys-N, and Lysarginase. As a result, we identify a total of 722 protein C-termini in E. coli, which is at least 66% more than the results using any single enzyme. Moreover, the favored enzyme and enzyme combination are discovered. Data are available via ProteomeXchange with identifier PXD004275.

Ginsenoside Rb1 protects against spinal cord ischemia-reperfusion injury in rats by downregulating the Bax/Bcl-2 ratio and caspase-3 and p-Ask-1 levels.

The aim of this study was to confirm the effects of ginsenoside Rb1 on neural cell apoptosis in the spinal cord of rats with spinal cord ischemia-reperfusion injury (SCII) and to explore its potential mechanisms. A total of 100 healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups: normal control (n = 10), sham-operated (n = 10), SCII model (n = 40), and ginsenoside Rb1-treated groups (n = 40). Basso, Beattie, Bresnahan (BBB) scale was used to examine rat hindlimb locomotor function. Nissl and Tunnel staining were used to observe neural cell injury and apoptosis, respectively, in the spinal cord of rats with SCII. Immunofluorescence staining was performed to detect the expression of Bax and Bcl-2. The levels of caspase-3 and phosphorylated Ask-1 (p-Ask-1) were detected by western blotting. Ginsenoside Rb1 prevented neural cell apoptosis in the spinal cord and improved hindlimb locomotor dysfunction of rats (P < .05). Moreover, SCII-induced upregulation of caspase-3 and p-Ask-1 levels and the Bax/Bcl-2 ratio were significantly decreased by ginsenoside Rb1 (P < .05). The protective effects of ginsenoside Rb1 on neural cells in the spinal cord of rats with SCII were mediated by the ginsenoside Rb1-induced downregulation of caspase-3 and p-Ask-1 levels and the Bax/Bcl-2 ratio.

Administration with curcumin alleviates spinal cord ischemia-reperfusion injury by regulating anti-oxidative stress and microglia activation-mediated neuroinflammation via Nrf2/NF-κB axis.

Spinal cord ischemia-reperfusion injury (SCII) ranks as the common complication after aortic surgery, usually leading to devastating post-operative paraplegia. Microglia over-activation and neuronal cell loss are key pathological features of SCII. Curcumin is involved in several I/R injuries. However, its underlying mechanism in SCII remains elusive. Here, curcumin attenuated oxygen and glucose deprivation/reoxygenation (OGD/R)-induced oxidative injury in PC12 neuronal cells by increasing cell viability, inhibiting cell apoptosis, lactate dehydrogenase, malondialdehyde levels, but elevating anti-oxidative superoxide dismutase and glutathione peroxidase levels. Furthermore, curcumin restrained OGD/R-evoked microglia M1 activation by decreasing microglia M1 polarization marker IBA-1 and iNOS transcripts. Moreover, the increased inflammatory cytokine levels of TNF-α and IL-6 in microglia under OGD/R conditions were suppressed after curcumin treatment. Importantly, neuronal cells incubated with a conditioned medium from OGD/R-treated microglia exhibited lower cell viability and higher apoptotic ratio, which were overturned when microglia were treated with curcumin. Intriguingly, curcumin could inhibit the activation of the NF-κB pathway by Nrf2 enhancement in OGD/R-treated PC12 cells and microglia. Notably, targeting Nrf2 signaling reversed the protective efficacy of curcumin against OGD/R-evoked oxidative insult in neuronal, microglia M1 activation, inflammatory response, and microglial activation-evoked neuronal death. In vivo, curcumin improved histopathologic injury and neurologic motor function in SCII rats and attenuated oxidative stress, microglia activation and neuroinflammation in spinal cord tissues, and activation of the Nrf2/NF-κB pathway. Thus, curcumin may alleviate SCII by mitigating I/R-evoked oxidative injury in neuron and microglia activation-induced neuroinflammation and neuron death through Nrf2/NF-κB signaling, supporting a promising therapeutic agent for SCII.

Identification of key genes involved in neural regeneration and the repairing effect of BDNF-overexpressed BMSCs on spinal cord ischemia-reperfusion injury in rats.

Bone marrow mesenchymal stem cells (BMSCs) can repair spinal cord ischemia-reperfusion injury (SCII); however, only a few BMSCs are usually located in the injured spinal cord. Since the brain-derived neurotrophic factor (BDNF) can promote neural development and maturation, we hypothesised that BDNF-overexpressed BMSCs can ameliorate SCII more effectively than BMSCs alone. To determine the effect of BDNF overexpression on SCII repair, BDNF-overexpressed BMSCs and BMSCs were transplanted into SCII rats. Our results revealed that BDNF-overexpressed BMSCs can better promote the recovery of damaged spinal cords than BMSCs alone. Gene chip detection of spinal cord tissues showed 803 differentially expressed genes in all groups. BTG anti-proliferation factor 2 (Btg2), FOS like 2 (Fosl2), early growth response protein 1 (Egr1), and serpin family E member 1 (Serpine1) were identified as key interrelated genes based on their expression trends, as validated via quantitative PCR and protein-protein interaction network analysis. A co-expression network was constructed to further explore the role of the candidate key genes using Pearson correlation analysis. Cluster 5 was identified as the key cluster using community discovery algorithms. Functional analysis of Cluster 5 genes revealed that this cluster was mainly involved in the stress-activated MAPK cascade, p38MAPK cascade, and apoptosis. Notably, Egr1 may play an important role in SCII repair as the top hub gene in Cluster 5. Therefore, the repair activity of transplanted BDNF-overexpressed BMSCs in SCII rats is better than that of BMSCs alone, which may be regulated by the interactions between Btg2, Fosl2, Egr1, Serpine1, and BDNF.

Ginsenoside Rg1 attenuates cerebral ischemia-reperfusion injury due to inhibition of NOX2-mediated calcium homeostasis dysregulation in mice.

Background: The incidence of ischemic cerebrovascular disease is increasing in recent years and has been one of the leading causes of neurological dysfunction and death. Ginsenoside Rg1 has been found to protect against neuronal damage in many neurodegenerative diseases. However, the effect and mechanism by which Rg1 protects against cerebral ischemia-reperfusion injury (CIRI) are not fully understood. Here, we report the neuroprotective effects of Rg1 treatment on CIRI and its possible mechanisms in mice. Methods: A bilateral common carotid artery ligation was used to establish a chronic CIRI model in mice. HT22 cells were treated with Rg1 after OGD/R to study its effect on [Ca2+]i. The open-field test and pole-climbing experiment were used to detect behavioral injury. The laser speckle blood flowmeter was used to measure brain blood flow. The Nissl and H&E staining were used to examine the neuronal damage. The Western blotting was used to examine MAP2, PSD95, Tau, p-Tau, NOX2, PLC, p-PLC, CN, NFAT1, and NLRP1 expression. Calcium imaging was used to test the level of [Ca2+]i. Results: Rg1 treatment significantly improved cerebral blood flow, locomotion, and limb coordination, reduced ROS production, increased MAP2 and PSD95 expression, and decreased p-Tau, NOX2, p-PLC, CN, NFAT1, and NLRP1 expression. Calcium imaging results showed that Rg1 could inhibit calcium overload and resist the imbalance of calcium homeostasis after OGD/R in HT22 cells. Conclusion: Rg1 plays a neuroprotective role in attenuating CIRI by inhibiting oxidative stress, calcium overload, and neuroinflammation.

Sevoflurane pretreatment regulates abnormal expression of MicroRNAs associated with spinal cord ischemia/reperfusion injury in rats.

BACKGROUND: Spinal cord ischemia/reperfusion injury (SCII) is one of the most serious spinal cord complications that stem from varied spine injuries or thoracoabdominal aortic surgery. Nevertheless, the molecular mechanisms underlying the SCII remain unclear. METHODS: Male Sprague-Dawley (SD) rats were randomly divided into 5 groups of sham, SCII 24 h, SCII 72 h, sevoflurane preconditioning SCII 24 h (SCII 24 h+sevo), and sevoflurane preconditioning SCII 72 h (SCII 72 h+sevo) group. We then analyzed the expression of differentially expressed micro RNAs (DEmiRNAs) in these groups and their target genes. Functional enrichment analysis of their target genes was further performed using Metascape software. The microRNA-messenger RNA-pathway (miRNA-mRNA-pathway) network and the sevoflurane-miRNA-mRNA-pathway integrative network were further constructed to explore the molecular mechanisms underlying SCII and neuroprotective effects of sevoflurane against SCII. Molecular docking was also performed to evaluate the interactions between hub targets and sevoflurane. Finally, the expression levels of miR-21-5p and its target genes [mitogen-activated protein kinase kinase 3 and protein phosphatase 1 regulatory subunit 3B (MAP2K3 and PPP1R3B)] were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses. RESULTS: We found that sevoflurane alters several miRNA expression following SCII at 24 and 72 h after reperfusion. It was shown that miR-221-3p, miR-181a-1-3p, and miR-21-5p were upregulated both at 24 and 72 h in the sevoflurane pre-treatment reperfusion groups. Functional enrichment analysis revealed that target genes for the above co-DEmiRNAs at 24 and 72 h in the SCII group with sevoflurane pretreatment participated in the mitogen-activated protein kinase (MAPK), ErbB, apoptosis, and transforming growth factor-beta (TGF-beta) signaling pathways. Both MAP2K3 and PPP1R3B were found to be common targets for sevoflurane and miRNA-mRNA-pathway (rno-miR-21-5p). It was shown that MAP2K3 regulates the MAPK signaling and the T cell receptor signaling pathways, whereas PPP1R3B regulates the ErbB signaling pathway. Molecular docking further revealed that sevoflurane strongly binds the MAP2K3 and PPP1R3B proteins. Compared to the sham group, SCII induced significant under-expression of miR-21-5p but upregulated PPP1R3B and MAP2K3 proteins; sevoflurane pretreatment increased the expression of miR-21-5p but decreased those of PPP1R3B and MAP2K3 proteins. CONCLUSIONS: In general, sevoflurane regulates the expression of several miRNAs following SCII. In particular, sevoflurane might protect against SCII via regulating the expression of miR-21-5p, its target genes (MAP2K3 and PPP1R3B), and related signaling pathways.

Spinal cord ischemia-reperfusion causes damage of neurocyte by inhibiting RAP2C.

OBJECTIVE: To explore the mechanism of RAP2C participating in spinal cord ischemia-reperfusion injury (SCII). MATERIALS AND METHODS: mRNA expressions of miR-204 and RAP2C were measured by qRT-PCR. Protein level of RAP2C was detected by Western blot. Cell apoptosis in AGE1.HN and PC12 cells was detected by Annexin V/propidium iodide assay. The effect of miR-204 knockdown or overexpression on RAP2C expression was observed by luciferase assay. RESULTS: In this experimental research, RAP2C expression in SCII group was lower than control group, while miR-204 expression was higher. In addition, RAP2C expression in AGE1.HN and PC12 cells under hypoxic condition was lower than control group, while miR-204 expression was higher. Hypoxia increased cell apoptosis, and overexpression of RAP2C reversed this effect. In cells treated with hypoxia and miRNA-204 inhibitor, hypoxia upregulated the expression of miR-204, while miRNA-204 inhibitor reversed this effect. Hypoxia downregulated the expression of RAP2C, while miR-204 inhibitor reversed this effect. Moreover, miR-204 could bind to 3'-UTR of RAP2C. And miR-204 inhibitor upregulated the activity and expression of RAP2C, while miR-204 mimic played the opposite role. Finally, hypoxia downregulated RAP2C expression, miR-204 inhibitor upregulated RAP2C expression, while Ad-sh-RAP2C inhibited RAP2C expression. What's more, hypoxia increased the apoptosis rate, miR-204 inhibitor decreased the apoptosis rate, while si-RAP2C increased the apoptosis rate. CONCLUSION: SCII causes increase of neurocyte apoptosis by inhibiting RAP2C via miR-204.

Down-regulated miR-448 relieves spinal cord ischemia/reperfusion injury by up-regulating SIRT1

MicroRNAs play a crucial role in the progression of spinal cord ischemia/reperfusion injury (SCII). The role of miR-448 and SIRT1 in SCII was investigated in this study, to provide further insights into prevention and improvement of this disorder. In this study, expressions of miR-448 and SIRT1 protein were determined by qRT-PCR and western blot, respectively. Flow cytometry was used to analyze cell apoptosis. The endogenous expression of genes was modulated by recombinant plasmids and cell transfection. Dual-luciferase reporter assay was performed to determine the interaction between miR-448 and SIRT1. The Basso, Beattie, and Bresnahan score was used to measure the hind-limb function of rat. The spinal cord ischemia reperfusion injury model of adult rats was developed by abdominal aorta clamping, and the nerve function evaluation was completed by motor deficit index score. In SCII tissues and cells treated with hypoxia, miR-448 was up-regulated while SIRT1 was down-regulated. Hypoxia treatment reduced the expression of SIRT1 through up-regulating miR-448 in nerve cells. Up-regulation of miR-448 induced by hypoxia promoted apoptosis of nerve cells through down-regulating SIRT1. Down-regulated miR-448 improved neurological function and hind-limb motor function of rats with SCII by up-regulating SIRT1. Down-regulated miR-448 inhibited apoptosis of nerve cells and improved neurological function by up-regulating SIRT1, which contributes to relieving SCII.