Calcyclin (S100A6) Attenuates Inflammatory Response and Mediates Apoptosis of Chondrocytes in Osteoarthritis via the PI3K/AKT Pathway.

OBJECTIVE: To clarify the regulatory effect of Calcyclin (S100A6) on chondrocytes apoptosis and its relationship with progression of osteoarthritis in an effort to explore potential therapeutic targets for osteoarthritis. METHOD: Immunofluorescence assay was produced to identify the rat chondrocyte sample and western blots assay was detected the expression changes of S100A6 between control group and osteoarthritis model which induced by interleukin-1ß. Adenovirus were transfected into the chondrocytes in vitro, in order to regulate the S100A6 expression. The influence of S100A6 on inflammatory reaction of osteoarthritis was detected by RT-PCR. Also, Caspase-3 activity assay and TUNEL assay were performed to evaluate the apoptosis changes. In addition, RT-PCR and western blots were performed to verify that S100A6 mediated the PI3K/AKT signaling pathway. Through the usage of pathway regulator, we detected S100A6 produced the effect by mediating the PI3K/AKT pathway. RESULTS: We determined the expression of S100A6 decreased in osteoarthritis model, the relative expression level in osteoarthritis model was about 0.5 fold compared with control group. Through adenovirus transfection we revealed that the inflammatory factors of osteoarthritis (interleukin-6 and matrix metalloproteinase-13) showed a negative correlation with the S100A6 expression. The relative expression level of interleukin-6 and matrix metalloproteinase-13 were 1.534 and 1.259 when S100A6 was up-regulated and the values were up to 2.445 and 2.074, respectively, when S100A6 was down-regulated. Also, the data verified the apoptosis could be reduced when the S100A6 was up-regulated and be activated when the S100A6 was down-regulated, the Caspase-3 activity was 16.512 U/µg and 24.45 U/µg respectively. Similar results were shown in TUNEL assay, the apoptosis index was 4.46% and 31.44%, respectively. Additionally, the results of polymerase chain reaction and western blots both demonstrated that the expression level of PI3K and AKT were increased when S100A6 was up-regulated, conversely the expression level of those two signal modules were reduced if the S100A6 was down-regulated. More importantly, the apoptosis triggered by S100A6 can be offset by the PI3K/AKT pathway inhibitor and activator (LY294002 and IGF-1), the values of Caspase-3 activity and apoptosis index became close to the untreated osteoarthritis group. The experimental results in this study were statistically significant. CONCLUSION: We investigated that Calcyclin (S100A6) relieved the inflammation and mediated the chondrocyte apoptosis through PI3K/AKT pathway and we confirmed that S100A6 might be an attractive therapeutic target.

Annexin A10 contributes to chronic constrictive injury-induced pain through activating ERK1/2 signalling in rats.

PURPOSE: The current study aims at investigating the downstream targets of spinal Annexin A10 in modulating neuropathic pain. MATERIALS AND METHODS: Paw withdrawal latency and paw withdrawal threshold were measured to evaluate the pain-associated behaviour in rats. The expression of spinal Annexin A10, phosphorylated-extracellular regulated kinase 1/2 and extracellular regulated kinase were detected by western blotting. The level of tumour necrosis factor-α and interleukine-1ß was tested by enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: Chronic constrictive injury caused pain hypersensitivity in rats, along with increased expression of spinal Annexin A10, phosphorylated-extracellular regulated kinase 1/2, tumour necrosis factor-α and interleukine-1ß in rats. Knockdown of spinal Annexin A10 suppressed the chronic constrictive injury-induced hyperalgesia, and inhibited the chronic constrictive injury-induced increased expression of phosphorylated-extracellular regulated kinase 1/2, tumour necrosis factor-α and interleukine-1ß in the spinal cord. Inhibition of spinal extracellular regulated kinase activation decreased the release of tumour necrosis factor-α and interleukine-1ß, but did not change the increased expression of Annexin A10 caused by chronic constrictive injury. CONCLUSIONS: Annexin A10 contributed to the development of neuropathic pain by activating spinal extracellular regulated kinase signalling and the subsequent release of tumour necrosis factor-α and interleukine-1ß in the spinal cord.

Propofol Prevents Hippocampal Neuronal Loss and Memory Impairment in Cerebral Ischemia Injury Through Promoting PTEN Degradation.

Neuroprotective effect of propofol against cerebral ischemia injury was widely investigated. However, its mechanisms remain unclear. Phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is supposed as a cell survival pathway, and phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a negative regulator of AKT phosphorylation. Whether PTEN was involved in the protective effect of propofol against cerebral ischemia injury was not elucidated. In this study, the function of PTEN in the acute phase of cerebral ischemia injury was investigated. Our data showed that propofol promoted the PTEN degradation in the acute phase of cerebral ischemia injury and concurrently activated AKT phosphorylation. The increase of ubiquitinated PTEN caused by cerebral ischemia injury were degraded in propofol-pretreated rats. Moreover, we evidenced that proteasome activity was stimulated in propofol-treated rats. These data pointed that PTEN degradation was facilitated in the acute phase after propofol treatment possibly through activating ubiquitin-proteasome system. Therefore, we applied PTEN inhibitor-bpV before cerebral ischemia injury. Like propofol, bpV pretreatment also mitigated cerebral ischemia injury-induced cell loss in CA1 region and memory impairment. Taken together, our data suggest that PTEN degradation is neuroprotective against cerebral ischemia injury and propofol facilitates PTEN degradation to prevent hippocampal neuronal loss and memory deficit in cerebral ischemia injury.

Piracetam prevents memory deficit induced by postnatal propofol exposure in mice.

Postnatal propofol exposure impairs hippocampal synaptic development and memory. However, the effective agent to alleviate the impairments was not verified. In this study, piracetam, a positive allosteric modulator of AMPA receptor was administered following a seven-day propofol regime. Two months after propofol administration, hippocampal long-term potentiation (LTP) and long-term memory decreased, while intraperitoneal injection of piracetam at doses of 100mg/kg and 50mg/kg following last propofol exposure reversed the impairments of memory and LTP. Mechanically, piracetam reversed propofol exposure-induced decrease of BDNF and phosphorylation of mTor. Similar as piracetam, BDNF supplementary also ameliorated propofol-induced abnormalities of synaptic plasticity-related protein expressions, hippocampal LTP and long-term memory. These results suggest that piracetam prevents detrimental effects of propofol, likely via activating BDNF synthesis.

Detrimental effects of postnatal exposure to propofol on memory and hippocampal LTP in mice.

Acute effects of propofol on memory and hippocampal long-term potentiation (LTP) in adult animals were reported. However, long-term effect of early postnatal application of propofol on memory was not totally disclosed. In this study, experiments were designed to verify the mechanisms underlying the long-term detrimental effects of propofol on memory and hippocampal synaptic plasticity. A consecutive propofol protocol from postnatal day 7 was applied to model anesthesia, long term memory and hippocampal synaptic plasticity were detected 2 months later. Our results showed that repeated propofol exposure in early phase affect the memory in the adult phase. Through recording the field excitatory postsynaptic potentials (fEPSPs) at Schaffer colletaral-CA1 synapses, both of basal synaptic transmission and hippocampal LTP were decreased after propofol application. While LTD induced by low frequency stimulation and 3,5-dihydroxyphenylglycine (3,5-DHPG) were not affected. Through analyzing the ultrastructure of dendrite in CA1 region, we found that propofol application decreased the spine density, which was consistent with the decrease of PSD-95 expression. In addition, p-AKT level was reduced after first propofol application. Intracerebroventricular injection of Akt inhibitor could mimic the propofol effects on basal synaptic transmission, hippocampal LTP and memory. Taken together, these results suggested that propofol possibly decreased AKT signaling pathway to restrict the spine development, finally leading to hippocampal LTP impairment and memory deficit.

Pten Inhibitor-bpV Ameliorates Early Postnatal Propofol Exposure-Induced Memory Deficit and Impairment of Hippocampal LTP.

Early postnatal propofol administration has potential detrimental effects on hippocampal synaptic development and memory. Therapeutic method is still lack due to unknown mechanisms. In this study, a 7-day propofol protocol was applied to model anesthesia in neonatal mice. Phosphatase and tensin homolog deleted on chromosome ten (Pten) inhibitor bisperoxovanadium (bpV) was pre-applied before propofol to study its potential protection. After propofol application, Pten level increased while phospho-AKT (p-AKT) (Ser473) decreased in dorsal hippocampus. Interestingly, i.p. injection of Pten inhibitor reversed the decrease of p-AKT. Two months after administration, basal synaptic transmission, hippocampal long-term potentiation (LTP) and long-term memory were reduced in propofol-administrated mice. By contrast, i.p. injection of Pten inhibitor at a dose of 0.2 mg/kg/day before propofol reversed the detrimental effects due to propofol application. Consistently, bpV injection also reversed propofol application-induced decrease of synaptic plasticity-related proteins, including p-CamKIIα, p-PKA and postsynaptic density protein 95. Taken together, our results demonstrate that bpV injection could reverse early propofol exposure-induced decrease of memory and hippocampal LTP. bpV might be a potential therapeutic for memory impairment after early propofol postnatal application.