Catalytic Mechanism and Heterologous Biosynthesis Application of Sesquiterpene Synthases.

Sesquiterpenes comprise a diverse group of natural products with a wide range of applications in cosmetics, food, medicine, agriculture, and biofuels. Heterologous biosynthesis is increasingly employed for sesquiterpene production, aiming to overcome the limitations associated with chemical synthesis and natural extraction. Sesquiterpene synthases (STSs) play a crucial role in the heterologous biosynthesis of sesquiterpene. Under the catalysis of STSs, over 300 skeletons are produced through various cyclization processes (C1-C10 closure, C1-C11 closure, C1-C6 closure, and C1-C7 closure), which are responsible for the diversity of sesquiterpenes. According to the cyclization types, we gave an overview of advances in understanding the mechanism of STSs cyclization from the aspects of protein crystal structures and site-directed mutagenesis. We also summarized the applications of engineering STSs in the heterologous biosynthesis of sesquiterpene. Finally, the bottlenecks and potential research directions related to the STSs cyclization mechanism and application of modified STSs were presented.

Two new sesquiterpenoids from plant endophytic fungus Flammulina velutipes.

Two new sesquiterpenoids, flammupin A (1) and flammupin B (2), along with two known compounds, enokipodin C (3) and 5,5'-dibuthoxy-2,2'-bifuran (4) were obtained from Flammulina velutipes, an endophytic fungus isolated from the roots of Caulophyllum robustum Maxim. The structures were elucidated by the combination of HR-ESI-MS, NMR, and ECD analyses. Compound 3 exhibited moderate to potent cytotoxicity against A549, HeLa, and SMMC-7721 cells with IC50 values ranged from 3.69 to 11.84 µM.

Ultrahigh-Throughput Screening of High-ß-Xylosidase-Producing Penicillium piceum and Investigation of the Novel ß-Xylosidase Characteristics.

A droplet-based microfluidic ultrahigh-throughput screening technology has been developed for the selection of high-ß-xylosidase-producing Penicillium piceum W6 from the atmospheric and room-temperature plasma-mutated library of P. piceum. ß-xylosidase hyperproducers filamentous fungi, P. piceum W6, exhibited an increase in ß-xylosidase activity by 7.1-fold. A novel ß-D-xylosidase was purified from the extracellular proteins of P. piceum W6 and designated as PpBXL. The optimal pH and temperature of PpBXL were 4.0 and 70 °C, respectively. PpBXL had high stability an acidic pH range of 3.0-5.0 and exhibited good thermostability with a thermal denaturation half-life of 10 days at 70 °C. Moreover, PpBXL showed the bifunctional activities of α-L-arabinofuranosidase and ß-xylosidase. Supplementation with low-dose PpBXL (100 µg/g substrate) improved the yields of glucose and xylose generated from delignified biomass by 36-45%. The synergism between PpBXL and lignocellulolytic enzymes enhanced delignified biomass saccharification, increased the Xyl/Ara ratio, and decreased the strength of hydrogen bonds.

Effects of dopamine transporter changes in the ventral tegmental area of the midbrain on cognitive function in aged rats.

The pathogenesis of Perioperative neurocognitive disorders (PND) is a synergistic effect of many factors. Up to now, the exact mechanism remains unclear. The dopamine pathway in the brain is one of the paths involved in the means of cognitive function. Therefore, the purpose of this study was to investigate the relationship between changes in dopamine transporters in the ventral tegmental area (VTA) of the midbrain and postoperative cognitive dysfunction in elderly rats. In this study, a mental dysfunction model in elderly rats was established after splenectomy under general anesthesia. Eighty male SD rats, aged 18-20 months, with a body mass of 300-500 g. Randomly divided into eight groups: Normal group (Normal, N) and Sham group (sham, S), Model 3 day group(PND, P3), Model 7 day group(PND, P7), Virus 3 days AAV·DAT·RNAi (AAV3), Virus 7 days AAV·DAT·RNAi (AAV7), Virus control for three days AAV·NC(NC3), Virus control for seven days AAV·NC(NC7). The results show that knockdown of dopamine transporter in the VTA region can significantly improve the cognitive dysfunction of elderly rats after surgery. These results suggest that dopamine transporter in the VTA region is involved in cognitive dysfunction in elderly rats. The effect of DAT changes in the VTA region on postoperative cognitive function in elderly rats may be related to the regulation of α-syn and Aß1-42 protein aggregation in the hippocampus.

Isoflurane post-conditioning contributes to anti-apoptotic effect after cerebral ischaemia in rats through the ERK5/MEF2D signaling pathway.

The mechanisms of brain protection during ischaemic reperfusion injury induced by isoflurane (ISO) post-conditioning are unclear. Myocyte enhancement factor 2 (MEF2D) has been shown to promote neural survival in a variety of models, in which multiple survival and death signals converge on MEF2D and modulate its activity. Here, we investigated the effect of MEF2D on the neuroprotective effects of ISO post-conditioning on rats after cerebral ischaemia/reperfusion (I/R) injury. Rats underwent middle cerebral artery occlusion (MCAO) surgery with ischaemia for 90 minutes and reperfusion for 24-48 hours. After MCAO, neurological status was assessed at 12, 24 and 48 hours by the Modified Neurological Severity Score (mNSS) test. The passive avoidance test (PAT) was used to assess cognition function. Histological and neuropathological evaluations were performed with HE staining and Nissl's staining, respectively. We measured the expression of MEF2D, ERK5, GFAP and caspase-3 by immunofluorescent staining and Western blotting, and TUNEL staining to assess the severity of apoptosis in hippocampal CA1 area. We found that MEF2D was involved in nerve protection after I/R injury, and post-treatment of ISO significantly promoted the phosphorylation of ERK5, increased MEF2D transcriptional activity, inhibited the expression of caspase-3 and played a role of brain protection.

Isoflurane post-conditioning attenuates cerebral ischemia/reperfusion injury by reducing apoptotic through activating the BMP7/SMAD signaling pathway in rats.

The expressions of different temporal patterns of bone morphogenetic proteins (BMPs) have changed after ischemic strokes, and ischemic preconditioning-induced neuroprotection was attenuated when BMP7 was inhibited. In the previous study, the neuroprotection of isoflurane postconditioning (ISPOC) against cerebral ischemia-reperfusion (I/R) injury has been addressed, with particular relevance to the role of BMP7. Consequently, in the present study, we continued to explore the mechanisms involved in the BMP7 signal mediated the neuroprotection of ISPOC. A rat model of the middle cerebral artery occlusion was used in this study. Rats were administered 1.5 % isoflurane, 60 min after 90 min of ischemia, followed by a 24 h reperfusion period. The 1.5 % ISPOC significantly ameliorated the cerebral infarct volumes, neurologic deficit scores, damaged neurons, and apoptotic neurons. Moreover, ISPOC unregulated the expressions of BMP7, p-Smad1/5/9, and p-p38. Whereas, the neuroprotective effect was weakened by LDN-193189 and SB203580, respectively, a BMP7/Smad1/5/9 and p38MAPK signaling pathway inhibitor. Furthermore, LDN-193189 downregulated the expression of p-p38. The present results of this study indicated that the neuroprotection of 1.5 % isoflurane postconditioning to cerebral ischemia-reperfusion injury is related to the activating of BMP7/Smad1/5/9 and p38MAPK signal pathway.

Isoflurane Postconditioning Upregulates Phosphorylated Connexin 43 in the Middle Cerebral Artery Occlusion Model and Is Probably Associated with the TGF-ß1/Smad2/3 Signaling Pathway.

AIM: Connexin 43 (Cx43) has been identified to be important for cerebral ischemia/reperfusion (I/R) injury as well as protection from it. This study was aimed at investigating the relationship between phosphorylated Cx43 (p-Cx43), transforming growth factor-ß1 (TGF-ß1 (TGF. METHODS: The middle cerebral artery occlusion (MCAO) model was induced in 96 male Sprague-Dawley rats, weighing 250-300 g. The rats were randomized into 12 groups, namely, sham, middle cerebral artery occlusion (MCAO)/I/R, I/R+1.5% ISPOC, I/R+LY2157299 (blocker of TGF-ß1 (TGF-ß1 (TGF-ß1 (TGF-ß1 (TGF. RESULTS: Neurological deficit scores, brain infarct volume, and damaged neurons in the I/R group significantly increased compared to those in the sham group (P < 0.05). However, in the ISPOC group, damage of the brain was significantly ameliorated (P < 0.05). However, in the ISPOC group, damage of the brain was significantly ameliorated (P < 0.05). However, in the ISPOC group, damage of the brain was significantly ameliorated (ß1 (TGF-P < 0.05). However, in the ISPOC group, damage of the brain was significantly ameliorated (ß1 (TGF-P < 0.05). However, in the ISPOC group, damage of the brain was significantly ameliorated (ß1 (TGF-ß1 (TGF-P < 0.05). However, in the ISPOC group, damage of the brain was significantly ameliorated (. CONCLUSION: Isoflurane postconditioning (ISPOC) may alleviate cerebral I/R injury through upregulating the expression of p-Cx43, and the TGF-ß1/Smad2/3 signaling pathway may be involved in the process.ß1 (TGF.

TGF-ß2/Smad3 Signaling Pathway Activation Through Enhancing VEGF and CD34 Ameliorates Cerebral Ischemia/Reperfusion Injury After Isoflurane Post-conditioning in Rats.

Evidence has shown the therapeutic potential of isoflurane (ISO) in cerebral stroke. The present study investigated the mechanism of ISO on vascular endothelial growth factor (VEGF) and CD34 expression in a rat model of stroke. Transient focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion for 24 h in rats. ISO was administered for 1.5 h when the reperfusion was initiated. Neurologic deficit scores, infarct volumes, HE staining, Nissl staining, and TUNEL staining were evaluated at 24 h after reperfusion. The levels of transforming growth factor (TGF)-ß2, Smad3, p-Smad3, VEGF, and CD34 proteins were detected by immunofluorescence (IF) staining and Western blot assay. Administration of ISO significantly reduced the neurologic deficit scores, infarct volumes, and damaged and apoptotic cells after cerebral ischemia/reperfusion (I/R) injury (P < 0.05). Meanwhile, ISO post-conditioning significantly increased the expression levels of TGF-ß2, p-Smad3, VEGF, and CD34 (P < 0.05), whereas the expression of Smad3 showed no difference (P > 0.05). However, Pirfenidone, a TGF-ß2 inhibitor, decreased the expression levels of TGF-ß2, p-Smad3, VEGF, and CD34 (P < 0.05). Moreover, the protective effects of ISO post-conditioning were negated by the inhibitor. The present study indicated that ISO attenuates brain damage by activating the TGF-ß2/Smad3 signaling pathway and increasing the protein expression of VEGF and CD34 in the rat MCAO model.

TGF-ß2 Induces Gli1 in a Smad3-Dependent Manner Against Cerebral Ischemia/Reperfusion Injury After Isoflurane Post-conditioning in Rats.

Isoflurane (ISO) post-conditioning attenuates cerebral ischemia/reperfusion (I/R) injury, but the underlying mechanism is incompletely elucidated. Transforming growth factor beta (TGF-ß) and hedgehog (Hh) signaling pathways govern a wide range of mechanisms in the central nervous system. We aimed to investigate the effect of the TGF-ß2/Smad3 and sonic hedgehog (Shh)/Glioblastoma (Gli) signaling pathway and their crosstalk in the hippocampus of rats with ISO post-conditioning after cerebral I/R injury. Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), 1.5 h occlusion and 24 h reperfusion (MCAO/R). To assess the effect of ISO after I/R injury, various approaches were used, including neurobehavioral tests, TTC staining, HE staining, Nissl staining, TUNEL staining, immunofluorescence (IF), qRT-PCR (quantitative real-time polymerase chain reaction) and Western blot. The ISO post-conditioning group (ISO group) received 1 h ISO post-conditioning when reperfusion was initiated, leading to lower infarct volumes and neurologic deficit scores, more surviving neurons, and less damaged and apoptotic neurons. IF staining, qRT-PCR and Western blot showed high expression levels of TGF-ß2, Shh and Gli1 in the hippocampal CA1 of the ISO group. Phosphorylated Smad3 (p-Smad3), Patched (Ptch), and Smoothed (Smo) were also increased at protein level in the ISO group, whereas total Smad3 expression did not change in all groups. When TGF-ß2 inhibitor, pirfenidone, or Smad3 inhibitor, SIS3 HCl, were administered, the expression levels of p-Smad3 and Gli1 were reduced, and surviving pyramidal neurons decreased. By contrast, the expression levels of TGF-ß2 and p-Smad3 did not change significantly after pre-injection of Smo inhibitor cyclopamine, but reduced the expression levels of Shh, Ptch, and Gli1. Moreover, Gli showed the lowest expression levels with pirfenidone combined with cyclopamine. These findings indicate that the TGF-ß and hedgehog signaling pathways mediate the neuroprotection of ISO post-conditioning after cerebral I/R injury, and crosstalk between two pathways at the Gli1 level.

Fluoxetine mitigating late-stage cognition and neurobehavior impairment induced by cerebral ischemia reperfusion injury through inhibiting ERS-mediated neurons apoptosis in the hippocampus.

Existing evidence from clinical and animal experiments all indicated that fluoxetine, selective serotonin-reuptake inhibitor (SSRI) and anti-depressant drug, has neuroprotection and improve functional outcomes after stroke. Endoplasmic reticulum stress (ERS) inducing apoptosis after cerebral ischemia reperfusion injury was demonstrated in our previous work. This trial was examined whether fluoxetine mitigates ERS-induced neuron apoptosis. Male sprague-dawley rats of cerebral ischemia reperfusion injury was produced via middle cerebral artery occlusion (MCAO) strategy, with ischemia for 90 min and reperfusion for 24 h. Experimental groups were divided into sham group, MCAO group, and fluoxetine group. 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used to evaluate cerebral infarct size. The expression of glucose-regulated protein 78 (GRP78), caspase-12, CHOP and caspase-3 were measured by Western blot and immunohistochemistry staining assays. Neurons apoptosis rate in the hippocampus was examined by the TUNEL assay. Neurobehavior examination was used to evaluate the motor function and passive avoidance test was used to assess cognition dysfunction. Fluoxetine treatment reduced the infarct size of rats after cerebral ischemia reperfusion injury. Furthermore, fluoxetine treatment decreased the expression of GRP-78, caspase-12, CHOP and caspase-3, and attenuated neurons apoptosis. Administration of fluoxetine promoted the damaged motor function. In the cognition test, after 4 days of fluoxetine treatment, cognition function of rats was improved. Fluoxetine treatment can mitigate cognition and neurobehavior impairment induced by cerebral ischemia reperfusion injury through inhibiting ERS-mediated neurons apoptosis in the hippocampus.

Isoflurane Post-conditioning Ameliorates Cerebral Ischemia/Reperfusion Injury by Enhancing Angiogenesis Through Activating the Shh/Gli Signaling Pathway in Rats.

Background: Stroke is the second leading cause of death worldwide. Angiogenesis facilitates the formation of microvascular networks and promotes recovery after stroke. The Shh/Gli signaling pathway is implicated in angiogenesis and cerebral ischemia-reperfusion (I/R) injury. This study aimed at investigating the influence of isoflurane (ISO) post-conditioning on brain lesions and angiogenesis after I/R injury. Methods: Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), 1.5 h occlusion and 24 h reperfusion (MCAO/R). The ISO post-conditioning group (ISO group) received 1 h ISO post-conditioning when reperfusion was initiated. Neurobehavioral tests, TTC staining, HE staining, Nissl staining, TUNEL staining, immunofluorescence (IF), immunohistochemistry (IH) and Western blot were performed to assess the effect of ISO after I/R injury. Results: ISO post-conditioning resulted in lower infarct volumes and neurologic deficit scores, higher rate of neurons survival, and less damaged and apoptotic cells after cerebral I/R injury in rats. Meanwhile, ISO post-conditioning significantly increased the expression levels of vascular endothelial growth factor (VEGF) and CD34 in the ischemic penumbra, relative to that in the Sham and I/R groups. However, cyclopamine, the specific inhibitor of the Sonic hedgehog (Shh) signaling pathway, decreased the expression levels of VEGF and CD34, and counteracted the protective effects of ISO post-conditioning against I/R injury in rats. Conclusions: ISO post-conditioning enhances angiogenesis in vivo partly via the Shh/Gli signaling pathway. Thus, Shh/Gli may represent new therapeutic targets for aiding recovery from stroke.

Wnt/ß-catenin signaling pathway contributes to isoflurane postconditioning against cerebral ischemia-reperfusion injury and is possibly related to the transforming growth factorß1/Smad3 signaling pathway.

AIM: The Wnt/ß-catenin signaling pathway plays an important role in ischemia-reperfusion(I/R) injury, and the transforming growth factor(TGF)-ß/Smad signaling pathway participates in the neuroprotection effect induced by isoflurane(ISO) postconditioning. In this study, we aimed to explore the role of the Wnt/|[beta]|-catenin ß-catenin signaling pathway in the neuroprotection effect induced by ISO postconditioning, and investigate the interaction of Wnt/ß-catenin and TGF-ß/Smad signaling pathway in this neuroprotection effect. METHODS: Cerebral I/R injury was established in Sprague-Dawley rats by using the middle cerebral artery occlusion (MCAO) model for 90 min followed by 24 h reperfusion. Postconditioning by inhalation of ISO was performed for 60 min after ischemia at the onset of reperfusion. Neurological deficit scoring, 2,3,5-triphenyl tetrazolium chloride staining and Nissl staining were adopted to evaluate brain injury. Apoptosis of the hippocampus and cortex neurons was detected by TUNEL staining. The expression levels of Wnt3a, GSK-3ß, ß-catenin, Cyclin D1, VEGF, Caspase 3, TGF-ß1, Smad3 and p-Smad3 were determined by immunofluorescence (IF) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Various targeted inhibitors were administered via intraperitoneal injection or lateral ventricle injection. RESULTS: In the cortex region, the neurological deficit score, infarct volumes and neuron apoptosis increased, and the expression level of the Wnt3a, GSK-3ß, ß-catenin, VEGF and Cyclin D1 decreased in the MCAO group compared with the Sham group. In the MCAO + ISO group, the neurological deficit score, infarct volumes and neuron apoptosis reduced significantly, the expression levels of Wnt3a, ß-catenin, VEGF and Cyclin D1 increased, while the expression level of GSK-3ß and Caspase 3 decreased relative to MCAO group. When Wnt inhibitor(DKK-1) was given in advance followed by ISO postconditioning, the neurological deficit score, infarct volumes, neuron apoptosis and the expression level of GSK-3ß and Caspase 3 increased. qRT-PCR and IF showed similar changes in the protein levels of all groups. However, the expression level of ß-catenin in nuclear and cytoplasm both decreased significantly after pre-injection with the TGF-ß1 inhibitor(LY2157299) and Smad3 inhibitor(SIS3), whereas the expression levels of TGF-ß1, Smad3 and p-Smad3 were almost unchanged. The expression levels of all the related proteins and morphological changes in the hippocampus region were consistent with that of the cortex. CONCLUSION: ISO postconditioning can reduce cerebral I/R injury by activating the Wnt/ß-catenin signaling pathway and may be related to the TGF-ß/Smad3 signaling pathway.

Isoflurane post-conditioning down-regulates expression of aquaporin 4 in rats with cerebral ischemia/reperfusion injury and is possibly related to bone morphogenetic protein 4/Smad1/5/8 signaling pathway.

AIM: Aquaporins (AQPs) are water-channels that play important roles in brain water homeostasis and cerebral edema induced by brain injury. This study aimed to investigate the relationship between AQP4, bone morphogenetic protein 4 (BMP4)/Smad1/5/8 signaling pathway and isoflurane post-conditiong, which has effects on brain edema in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: Cerebral I/R injury was induced in rats by using the middle cerebral artery occlusion (MCAO) model for 90min, followed by 24h of reperfusion. Isoflurane post-conditioning (ISO) group received 90min ischemia and underwent 1.5% isoflurane post-conditioning for 60min after initiating reperfusion. Neurobehavior, brain water content, thionine staining and 2, 3, 5-triphenyl tetrazolium chloride staining were evaluated to measure levels of brain edema and damage. Expressions of AQP4, BMP4, Smad1/5/8 and phosphorylated Smad1/5/8 were detected by using Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence (IF) staining. RESULTS: Compared with the Sham group, neurological behavior score, brain infarct volume and water content of MCAO model rats increased with reperfusion injury. However, in the ISO group, cell edema and damage of brain was significantly ameliorated (P<0.01). qRT-PCR showed less AQP4 mRNA expression in the hippocampal tissue of the ISO group than in the I/R group (P<0.01). Western blot and immunofluorescence results showed similar changes in protein levels of both groups. Related protein expressions showed expressions of BMP4 and Smad1/5/8 increased in the ISO group (P<0.01), whereas total Smad1/5/8 expression didn't change in all groups. When BMP4 inhibitor (LDN193189) was injected, expression levels of AQP4 increased and neuronal density decreased (P<0.05). By contrast, expression levels of BMP4 did not change significantly after pre-injection of AQP4 inhibitor (TGN020) (P>0.05), but neuronal density increased (P<0.05). CONCLUSION: Isoflurane post-conditioning may inhibit occurrence of brain edema and reduce cerebral I/R injury through down-regulating expression of AQP4, This process may be related to the activation of BMP4/Smad1/5/8 signaling pathway.

Effect of ephedrine combined with bupivacaine on maternal hemodynamic and spinal nerve block in cesarean delivery.

The aim of the study was to investigate the effects of ephedrine intrathecal injection on maternal thermodynamics and spinal nerve block in cesarean section. A total of 107 patients undergoing elective cesarean section under combined spinal epidural anesthesia were randomly divided into two groups: group E: Bupivacaine (6 mg) combined with ephedrine (15 mg) and group C: Bupivacaine (6 mg). Systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), the incidence of hypotension, hypertension, tachycardia, nausea and vomiting were all recorded. The umbilical vein blood gases and noradrenaline, as well as maternal peripheral venous blood were analyzed. The time of high-quality spinal block was also recorded. Postoperatively, 1 day, 2 days bedside follow-up and 7, 21, 35 days telephone follow-up were carried out to determine any neurological deficit maternal performance. SBP and DBP decreased in group C but not in group E at 3 min after spinal anesthesia and 1 min after delivery (P<0.05). From 3 min post-anesthesia to the end of surgery, the HR levels in the C group were significantly higher than those of the E group (P<0.05). The incidence of hypotension, tachycardia, nausea and vomiting and neonate acidosis was low in group E compared with group C (P<0.05). The time of high-quality spinal block in group E was longer than that of group C (P<0.05). After spinal anesthesia, the concentration of noradrenaline from maternal peripheral venous blood was low in group C but high in group E compared with before spinal anesthesia (P<0.05). Ephedrine combined with low-dose bupivacaine intrathecal injection effectively maintained maternal hemodynamics and prolonged the time of sensory spinal block.

Effects of activin A and its downstream ERK1/2 in oxygen and glucose deprivation after isoflurane-induced postconditioning.

BACKGROUND: Isoflurane postconditioning (ISPOC) plays a neuroprotection role in the brain. Previous studies confirmed that isoflurane postconditioning can provide better protection than preconditioning in acute hypoxic-ischemic brain damage, such as acute craniocerebral trauma and ischemic stroke. Numerous studies have reported that activin A can protect rat's brain from cell injury. However, whether activin A and its downstream ERK1/2 were involved in isoflurane postconditioning-induced neuroprotection is unknown. METHODS: A total of 80 healthy Sprague-Dawley rats weighing 50-70g were randomly divided into 10 groups of 8: normal control, oxygen and glucose deprivation (OGD), 1.5% ISPOC, 3.0% ISPOC, 4.5% ISPOC, blocker of activin A (SB431542), blocker of ERK1/2 (U0126), 3.0% ISPOC+SB431542, 3.0% ISPOC+U0126, and vehicle (dimethyl sulfoxide(DMSO)) group. Blockers (SB431542 and U0126) were used in each concentration of isoflurane before OGD. Hematoxylin-eosin staining, 2,3,5-triphenyl tetrazolium chloride staining, and propidium iodide (PI) staining were conducted to assess the reliability in the brain slices. Immunofluorescence, Western blot, and quantitative real-time PCR(Q-PCR) were performed to validate the protein expression levels of activin A, Smad2/3, P-Smad2/3, ERK1/2, and phosphorylation ERK1/2 (P-ERK1/2). RESULTS: The number of damaged neurons and mean fluorescence intensity(MFI) of PI staining increased, but formazan generation, expression levels of activin A and P-ERK1/2 protein, and mRNA synthesis level of activin A decreased in the OGD group compared with the normal control group (p<0.05). The number of damaged neurons and MFI of PI staining decreased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A increased significantly in the 1.5% ISPOC and 3.0% ISPOC groups (p<0.05) compared with the OGD group. The result in the 4.5% ISPOC group, was completely opposite to the 1.5% ISPOC and 3.0% ISPOC groups. The number of damage neuron and MFI of PI staining increased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A decreased in the 4.5% ISPOC group. However, the expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A in the 4.5% ISPOC group were higher than the OGD group (p<0.05). The other results were compared between the SB431542 group/the U0126 group and 3.0% ISPOC group. The MFI of PI staining increased, but the expression levels of activin A, P-Smad2/3, and P-ERK1/2 decreased (p<0.05). The expression level of ERK1/2 protein in all groups exhibited no change (p>0.05). CONCLUSION: Results of this study showed that 3.0% concentration of isoflurane postconditioning provided better neuroprotection than 1.5% and 4.5% concentrations of isoflurane. Activin A/Smad 2/3 and activin A/ERK1/2 signaling pathway may be involved in ISPOC-induced neuroprotection.

Transforming growth-beta 1 contributes to isoflurane postconditioning against cerebral ischemia-reperfusion injury by regulating the c-Jun N-terminal kinase signaling pathway.

AIM: Cerebral ischemia-reperfusion (I/R) injury is a devastating complication in the perioperative period. Transforming growth factor beta (TGF-ß) is a key protein that can participate in the repair and control process responses after I/R injury. Isoflurane is widely used in neurosurgery. Previous studies have shown that isoflurane preconditioning plays an important role in neuroprotection. However, the effects of isoflurane postconditioning on cerebral I/R injury have not yet been elucidated. In the present study, we evaluated the protective effect of isoflurane postconditioning against cerebral I/R injury and investigated the role of the TGF-ß signaling pathway and the downstream c-Jun N-terminal kinase (JNK) signaling pathway in neuroprotective mechanism. In particular, the JNK signaling pathway emerges as a possible target for brain repair after stroke. METHODS: Cerebral I/R injury was produced in SD rat by using the middle cerebral artery occlusion model for 90 min, followed by 24h reperfusion. Postconditioning by inhalation of isoflurane was performed at different concentrations (1.5%, 3.0%, and 4.5%) for 1h after ischemia at the starting time point of reperfusion. The protective effect was tested by neurological deficit scoring with 2,3,5-triphenyl tetrazolium chloride and propidium iodide (PI) staining. Apoptosis of CA1 cells in the hippocampus was detected by TUNEL method. Expression levels of TGF-ß1, Smad 2/3, p-Smad2/3, JNK, and p-JNK were determined by immunostaining and Western blot. RESULTS: Postconditioning by isoflurane at 1.5% and 3.0% concentrations significantly decreased the neurobehavioral deficit scores and infarct volume compared with the I/R group, but no significant difference in neurobehavioral deficit score was detected between the I/R and 4.5% isoflurane postconditioning groups. Additionally, 1.5% isoflurane postconditioning decreased the numbers of PI-positive cells at 24h after reperfusion compared with the I/R group. TGF-ß1 and p-Smad2/3 protein gradually increased after I/R injury, with the highest values observed in the 1.5% and 3% isoflurane postconditioning groups. For Smad2/3 protein expression, no differences existed among all groups. After inducing the TGF-ß/SMAD3 signaling pathway specific blocker (LY2157299), the neurological deficit scores increased, infarct volumes enlarged, apoptosis increased, and PI-positive CA1 cells in the hippocampus also increased. The expression levels of TGF-ß1 and p-Smad2/3 proteins were downregulated. During the pre-injection of LY2157299, the expression levels of TGF-ß1 and p-Smad2/3 decreased significantly, but compared with the sham group, the expression level of p-JNK significantly increased. When the injection of LY2157299 was abolished, the expression of p-JNK significantly decreased. The expression levels of p-JNK and TGF-ß1 significantly decreased when LY2157299 and SP600125 were injected simultaneously. However, the protective effect mediated by SP600125 completely disappeared, and the role of LY2157299 became dominant. Compared with the sham group, the expression of TGF-ß1 was almost unchanged by the injection of SP600125 alone, but the expression of p-JNK significantly decreased. CONCLUSIONS: Up to 1.5% isoflurane can upregulate the expression of TGF-ß1 and downregulate that of p-JNK, which significantly mitigated I/R injury, leading to cerebral injury. However, this protective effect was abrogated when the TGF-ß1 signaling pathway was blocked by LY2157299. Overall, the present results provided valid evidence to demonstrate that TGF-ß1 contributes to isoflurane postconditioning against cerebral I/R injury by inhibiting the JNK signaling pathway.