Effect of anesthesia on the expression of clock gene Clock and Bmal1 in New Zealand rabbits. / éº»é†‰è¯ç‰©å¯¹æ–°è¥¿å °å ”é’ŸåŸºå› Clock和Bmal1表达的影响.

OBJECTIVES: To evaluate the effect of anesthetic drugs on the expression of circadian gene Clock and Bmal1 in the brain of New Zealand rabbits, and to explore their change pattern. METHODS: A total of 90 New Zealand rabbits were randomly divided into 5 groups (n=18 in each group): A normal saline group (1 mL/h saline, group S), a propofol group [600 µg/(kg·min-1) propofol, 1 mL/h, group P], a 10% lipid group (1 mL/h lipid, group F), a dexmedetomidine group [1 µg/(kg·min-1) dexmedetomidine, 1 mL/h, group D), a sevoflurane (SEV) group (2.5% SEV, SEV group). Inhaled and intravenous anesthetic drugs were stopped after 24 h. Experimental animals were killed at 0, 24, and 72 h after anesthesia, and their brain tissues were isolated. Western blotting was performed to measure the Clock and Bmal1 protein expression in the brain of rabbits. RESULTS: At 0 and 24 h after anesthesia, compared with the group S, the levels of Clock and Bmal1 proteins were decreased significantly in the group P, the group D, and the group SEV (all P<0.05). At 72 h after anesthesia, compared with the group S, the levels of Clock and Bmal1 proteins showed no significant changes in the group P, the group D, and the SEV group (all P>0.05). Compared with the group S, the levels of Clock and Bmal1 proteins at all time points showed no significant changes in the group F (all P>0.05). CONCLUSIONS: Anesthetic SEV, propofol, and dexmedetomidine can inhibit the expression of clock gene Clock and Bmal1 protein in the brain fissues of the New Zealand rabbits, and the suppression effect continues for at least 24 h after anesthesia, whereas the suppression decreases significantly at 72 h after anesthesia.

The Key Target and Molecular Mechanism of the Volatile Component of Scutellaria baicalensis Georgi in Acute Lung Injury Based on Network Pharmacology.

Ethnopharmacological relevance: Scutellaria baicalensis georgi is one of the most widely studied TCMs; its effects in ALI have been studied in a large number of experiments, and the efficacy of volatile oil from TCM remains to be studied. Aim: The volatile component of Scutellaria baicalensis georgi was selected to act on the key target of acute lung injury and was preliminarily studied for its specific molecular mechanism. Methods: The volatile active substances of Scutellaria baicalensis georgi were extracted by GC-MS, and the active ingredients related with the occurrence and development of acute lung injury were searched and matched by the TCMSP database. The pharmacologic data and analysis platform of TCM were used to retrieve and screen for the volatile active components and the possible therapeutic targets of Scutellaria baicalensis georgi. In addition, acute lung injury was searched in the disease target database to identify the corresponding disease target proteins, thereby establishing a protein-protein interaction network. Finally, the effects of wogonin on the apoptotic and inflammatory factors in the acute lung injury cell model were analyzed experimentally. Results: We identified 100 candidate targets and successfully constructed a complex target network. The targets identified by the above gene enrichment analysis played important roles in the autoimmune disease cell cycle apoptosis and related signaling pathways. The KEGG pathway analysis showed that most of the target genes were involved in the inflammatory response regulation of the TRP, PI3K-Akt, and IL-17 signaling pathways. The participation of wogonin in the specific regulatory pathways of PI3K-Akt signaling and IL-17 signaling was verified through experiments. In the lung-injured cell model, the results showed that wogonin inhibited the apoptosis of injured lung cells by inhibiting the expression of BAD gene and the activation of cleaved caspase-3 gene while increasing Bcl-2 expression. In addition, wogonin inhibited the expression of the abovementioned inflammatory factors and further inhibited the inflammatory response in the lung injury cells. Conclusion: The results of pharmacological network analysis can predict and explain the regulation mechanism of multi-target and multi-pathway of TCM components. This study identified the potential target and important pathway of wogonin in regulating acute lung injury. At the same time, the accuracy of network pharmacological prediction is also preliminarily verified by molecular biology experiment.

Dexmedetomidine alleviates sleep-restriction-mediated exaggeration of postoperative immunosuppression via splenic TFF2 in aged mice.

Major abdominal procedures could induce dysfunction in the immune system and lead to postoperative immunosuppression. Sleep dysfunction is associated with impaired immune activity. However, the effects of postoperative sleep dysfunction on postoperative immune function remain unclear. In this study, we found that sleep-restriction (SR) after surgery increased the spleen weight and the percentage of myeloid-derived suppressor cells (MDSCs) in the spleen, and inhibited splenic CD8+ T cells activity, which was via inhibiting subdiaphragmatic vagus nerve (SVN)-mediated trefoil factor 2 (TFF2) expression in the spleen of aged mice. Dexmedetomidine could alleviate SR-induced these changes via modulating gut microbiota, which acted through SVN. Moreover, we showed essential roles of splenic TFF2 in attenuating SR-induced reduced protective ability against Escherichia coli (E. coli) pneumonia, increased expression of IL-4 and IL-13 in the lung and M2 polarization of alveolar macrophages (AMs), and decreased phagocytic activity of AMs. Dexmedetomidine improved SR-induced reduced protective ability against E. coli pneumonia via splenic TFF2, and subsequently decreasing IL-4 and IL-13 expression in the lung via modulating gut microbiota/SVN, increasing the compromised phagocytic activity of AMs, and ultimately decreasing M2 polarization of AMs. Taken together, dexmedetomidine-induced increase in splenic TFF2 expresssion could alleviate SR-induced exaggeration of postoperative immunosuppression.

Prediction of immune factors and signaling pathways in lung injury induced by LPS based on network analysis.

OBJECTIVE: To construct a regulatory network involved in acute lung injury, so as to provide a new theoretical basis and research ideas for studying the relationship between inflammatory factors and immune proteins to collectively regulate the occurrence of acute lung injury. METHOD: By using Meta-analysis, GO, KEGG and other methods notarized and constructed the regulatory network pathways of cytokine cascade and lung injury induced by LPS. RESULTS: The result of Meta-analysis showed that the correlation between CD14, TNF-α, IL-6 gene and acute lung injury was statistically significant. GO analysis and KEGG analysis showed that acute lung injury contained CD14, TNF-α, IL-6 and other involved factors in the induced process of LPS, these inflammatory factors and immune proteins jointly regulate the process of disease development. CONCLUSION: CD14 receptor is an important receptor involved in mediating LPS-activated cells, and is a high-affinity LPS receptor. LPS stimulates inflammatory effector cells to bind to LPS receptor- CD14 to activate intracellular signal cascade. Direct or indirect involvement of pathogenic factors enable cytokine caused by induction form a particularly complex network of cytokine regulatory pathways, of which the inflammatory factors TNF-α and IL-6 are simultaneously involved in LPS-mediated and CD14-mediated cytokine cascades.

Enriched housing promotes post-stroke neurogenesis through calpain 1-STAT3/HIF-1α/VEGF signaling.

Enriched environment (EE) has been shown to promote neurogenesis and functional recovery after ischemic stroke. However, the underlying molecular mechanisms are not fully understood. In this study, C57BL/6 mice underwent middle cerebral artery occlusion (60 min) followed by reperfusion, after which mice were housed in either standard environment (SE) or EE and allowed to survive for 3, 4, 6 or 10 weeks. Ipsilateral subventricular zone (SVZ) or striatum cells were dissociated from ischemic hemispheric brains of enriched mice at 14 days post-ischemia (dpi) and cultured in vitro. Our data showed that post-ischemic EE inhibited calpain 1 activity, and increased the expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in the ischemic hemisphere of enriched mice at 21 dpi. Calpain 1-specific inhibitor PD151746 further increased p-STAT3 expression and augmented the promoting effects of EE on post-stroke SVZ neural precursor cells (NPCs) proliferation and functional recovery. EE also increased the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in the ischemic hemisphere at 21 dpi. Inhibition of the JAK/STAT3 pathway with AG490 decreased the expression of HIF-1α and VEGF. Furthermore, inhibition of HIF-1α with 2-methoxyestradiol robustly abolished EE-induced elevation of VEGF l expression. Furthermore, VEGF-A promoted the production and secretion of high mobility group box-1 protein (HMGB1) from reactive astrocytes in vitro. Culture supernatant from reactive astrocytes treated with VEGF-A promoted the proliferation and differentiation of NPCs. Glycyrrhizin reversed the promoting effects of EE on post-stroke neurorepair and functional recovery in vivo. Taken together, our data indicate that EE promotes post-stroke functional recovery through the inhibition of calpain 1 activity, and subsequent STAT3-HIF-1α-VEGF-mediated neurogenesis.

Long non-coding RNA CCAT1 modulates neuropathic pain progression through sponging miR-155.

Neuropathic pain is caused by dysfunction or primary injury of the somatosensory nervous system. Long noncoding RNAs (lncRNAs) play important roles in the development of neuropathic pain. However, the effects of lncRNA colon cancer associated transcript-1 (CCAT1) in neuropathic pain have not been reported. The model of bilateral sciatic nerve chronic constriction injuries (bCCI) is regarded as long-lasting mechanical hypersensitivity and cold allodynia, which is the representative symptom in the human subjects suffering from the neuropathic pain. In this study, we found that CCAT1 expression was decreased in the spinal dorsal horn, dorsal root ganglion (DRG), hippocampus, and anterior cingulate cortex (ACC) of rats with bCCI. The rats of bCCI presented the cold allodynia after the 14th day of postoperation. We furtherly showed that lncRNA CCAT1 decreased miR-155 expression and enhanced Serum and glucocorticoid regulated protein kinase 3 (SGK3) expression in the NGF-differentiated PC12 cell. We found that miR-155 expression was increased in the spinal dorsal horn, DRG, hippocampus, and ACC of rats with bCCI injuries. However, SGK3 expression was downregulated in the spinal dorsal horn, DRG, hippocampus, and ACC of rats with bCCI injuries. Moreover, lncRNA CCAT1 overexpression could alleviate the pain thresholds and inhibited expression of SGK3 could rescue this effect. In conclusion, these results suggested the crucial roles of CCAT1 and SGK3 in the neuropathic pain.

Alpha7 nicotinic acetylcholine receptor activation attenuated intestine-derived acute lung injury.

BACKGROUND: Intestinal ischemia-reperfusion (IIR) could lead to acute lung injury, associated with severe alveolar epithelial cells inflammatory and oxidative injury. Alpha7 nicotinic acetylcholine receptor (α7nAChR) is an essential component of the cholinergic anti-inflammatory pathway. The aim of this study was to investigate the important role of α7nAChR on the lung subjected to IIR. METHODS: Thirty-two Sprague-Dawley rats were randomly divided into four groups (n = 8 in each): sham group (group S), model group (group M), α7nAChR agonist PNU-282987-treated group (group PNU), and specific α7nAChR antagonist methyllycaconitine-treated group (group MLA). Intestinal IR damage was induced by clamping the superior mesenteric artery for 75 min, followed by a 120-min reperfusion. All rats were killed at 2 h after release of the clamps. The histologic examination of lungs was made, and lung water content was detected. Expression levels of malondialdehyde, tumor necrosis factor alpha, interleukin-6, and superoxide dismutase activity of the lungs were detected. Additionally, expression level of toll-like receptor (TLR)4 and nuclear factor-kappaB (NF-κB p65) in the nucleus of lung tissue and apoptosis-related protein (Bax, Bcl-2, and cleaved-caspase3) were detected using Western blot. RESULTS: Lungs were damaged after intestine IR, manifested by higher lung water content, histologic score, concentrations of interleukin-6, tumor necrosis factor alpha, and malondialdehyde of group M than those of group S, accompanied with decreased superoxide dismutase activity (P < 0.05). PNU treatment could significantly improve the pulmonary function of rats subjected to IIR. These effects of activation of α7nAChR were associated with suppression of TLR4/NF-κB pathway and subsequent reduction of apoptosis-related protein. However, MLA treatment aggravated lung injury. CONCLUSIONS: α7nAChR plays a role in acute lung injury induced by IIR via attenuating lung oxidative stress and inflammation through suppression of TLR4/NF-κB pathway, resulting in reduction of apoptosis in the lung.

Propofol alleviates acute lung injury following orthotopic autologous liver transplantation in rats via inhibition of the NADPH oxidase pathway.

Acute lung injury (ALI) induced by liver transplantation is detrimental to patient survival, and therapeutic strategies remain limited. Thus, the protective effects of propofol, a commonly used anesthetic with antioxidative and anti­inflammatory properties, were investigated in the present study on ALI induced by orthotopic autologous liver transplantation (OALT). The protective mechanism of propofol was determined to be associated with the inhibition of NADPH oxidase, by comparing its effects with the positive controls apocynin (AP; an NADPH oxidase inhibitor) and N­acegysteine (NAC; a scavenger of reactive oxygen species). The results demonstrated that two proteins (p47phox and gp91phox) of the NADPH oxidase system presented increased expression in rats with ALI induced by OALT, thus leading to increased activation of the oxidative stress and inflammatory reactions. Preconditioning with NAC or AP eliminated this increase, suggesting that antioxidative treatment, particularly with inhibitors of NADPH oxidase, is a promising protective strategy against ALI induced by OALT. Propofol preconditioning at a high (100 mg/kg) or low (50 mg/kg) dose promoted similar protective effects, with the high­dose propofol producing a more marked effect than the low dose. The results suggested that propofol may protect against ALI induced by OALT, the mechanism of which may involve a reduced oxidative stress and inflammatory reaction mediated by NADPH oxidase inhibition.

Propofol activation of the Nrf2 pathway is associated with amelioration of acute lung injury in a rat liver transplantation model.

OBJECTIVE: This study aimed to investigate whether propofol pretreatment can protect against liver transplantation-induced acute lung injury (ALI) and to explore whether Nrf2 pathway is involved in the protections provided by propofol pretreatment. METHOD: Adult male Sprague-Dawley rats were divided into five groups based on the random number table. Lung pathology was observed by optical microscopy. Lung water content was assessed by wet/dry ratio, and PaO2 was detected by blood gas analysis. The contents of H2O2, MDA, and SOD activity were determined by ELISA method, and the expression of HO-1, NQO1, Keap1, and nuclear Nrf2 was assayed by western blotting. RESULTS: Compared with saline-treated model group, both propofol and N-acetylcysteine pretreatment can reduce the acute lung injury caused by orthotopic autologous liver transplantation (OALT), decrease the lung injury scores, lung water content, and H2O2 and MDA levels, and improve the arterial PaO2 and SOD activity. Furthermore, propofol (but not N-acetylcysteine) pretreatment especially in high dose inhibited the expression of Keap1 and induced translocation of Nrf2 into the nucleus to further upregulate the expression of HO-1 and NQO1 downstream. CONCLUSION: Pretreatment with propofol is associated with attenuation of OALT-induced ALI, and the Nrf2 pathway is involved in the antioxidative processes.

Knockdown of myeloid differentiation protein-2 reduces acute lung injury following orthotopic autologous liver transplantation in a rat model.

BACKGROUND: Acute lung injury (ALI) is a serious complication that commonly occurs during orthotopic liver transplantation (OLT). Toll-like receptor 2/4 (TLR2/4) are the main membrane receptors that respond to inflammatory stimuli and mediate NF-kappa B (NF-κB) signal pathway. We previously showed that TLR2/4 expression on monocytes and serum cytokine levels were increased in patients with ALI induced by OLT. Myeloid differentiation protein-2 (MD-2) expresses the functional domains that combines TLRs and play a key regulatory role in TLRs activation. Therefore, we hypothesized that blocking MD-2 would inhibit the TLR2/4-mediated inflammatory response and lessen ALI induced by liver transplantation. METHOD: Thirty-two Sprague Dawley (SD) rats were randomly divided into four groups. One group received a sham operation (Group S), and the other three groups underwent orthotopic autologous liver transplantation (OALT) 48 h after intratracheal administration of saline (Model group; Group M), non-targeting siRNA (negative siRNA control group; Group NC) or siRNA against MD-2 (intervention group; Group RNAi). Lung pathology, lung water content, PaO2, and expression levels of MD-2, TLR2/4, NF-κB, TNF-α, IL-1ß and IL-6 were assessed 8 h after OALT. RESULTS: In Groups M and NC, OALT produced marked lung pathology with decreased PaO2 levels and increased MD-2, TLR2/4 gene and protein expression levels. Furthermore, the nuclear translocation of the NF-κB P65 subunit, was increased, as were lung concentrations of TNF-α, IL-1ß and IL-6. The pathology of ALI and the severity of the above biochemical changes induced by OALT were significantly reduced in the group treated with MD-2 siRNA. CONCLUSION: MD-2 gene knock-down attenuated the increase in TLR2/4 activation and reduced ALI after OALT.