Effect of scutellarin on BV-2 microglial-mediated apoptosis in PC12 cells via JAK2/STAT3 signalling pathway.

Previous studies have shown that scutellarin inhibits the excessive activation of microglia, reduces neuronal apoptosis, and exerts neuroprotective effects. However, whether scutellarin regulates activated microglia-mediated neuronal apoptosis and its mechanisms remains unclear. This study aimed to investigate whether scutellarin can attenuate PC12 cell apoptosis induced by activated microglia via the JAK2/STAT3 signalling pathway. Microglia were cultured in oxygen-glucose deprivation (OGD) medium, which acted as a conditioning medium (CM) to activate PC12 cells, to investigate the expression of apoptosis and JAK2/STAT3 signalling-related proteins. We observed that PC12 cells apoptosis in CM was significantly increased, the expression and fluorescence intensity of the pro-apoptotic protein Bax and apoptosis-related protein cleaved caspase-3 were increased, and expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) was decreased. Phosphorylation levels and fluorescence intensity of the JAK2/STAT3 signalling pathway-related proteins JAK2 and STAT3 decreased. After treatment with scutellarin, PC12 cells apoptosis as well as cleaved caspase-3 and Bax protein expression and fluorescence intensity decreased. The expression and fluorescence intensity of Bcl-2, phosphorylated JAK2, and STAT3 increased. AG490, a specific inhibitor of the JAK2/STAT3 signalling pathway, was used. Our findings suggest that AG490 attenuates the effects of scutellarin. Our study revealed that scutellarin inhibited OGD-activated microglia-mediated PC12 cells apoptosis which was regulated via the JAK2/STAT3 signalling pathway.

Gastrodin exerts protective effects in reactive TNC1 astrocytes via regulation of the Notch signaling pathway.

BACKGROUND: Gastrodin (GAS) has been proven to play a therapeutic role in a variety of neurological diseases by affecting activated astrocytes, however, the underlying mechanisms have not been fully illustrated. This study aimed to investigate if GAS exerts the neuroprotective effect through regulating the Notch signaling pathway involved in reactive astrocytes. METHODS: Astrocyte cell lines (TNC1 cells) were cultured in vitro. The hypoxic-ischemic cell model was prepared using the oxygen-glucose deprivation (OGD) method, GAS's pretreatment concentration was 0.34 mM, intervention for 1 hour. Cell counting kit-8 (CCK-8) assay, Transwell migration assay, immunofluorescent staining (double staining), and Western blotting were used to observe the effects of OGD or GAS interference on the function of astrocytes, and the changes of key protein expressions in the Notch signaling pathway were analyzed. RESULTS: GAS had no obvious toxic effect on TNC1 astrocytes under physiological conditions. Following OGD, GAS can not only improve cell viability and migration, but also regulate the production of inflammatory mediators. We also found that OGD significantly increased the expression of key proteins related to the Notch signaling pathway, Notch-1, intracellular Notch receptor domain (NICD), recombining binding protein suppressor of hairless (RBP-JK), transcription factor hairy and enhancer of split-1 (Hes-1) in TNC1 astrocytes, which was significantly inhibited by GAS. In addition, GAS inhibited the OGD-induced expression of TNC1 astrocyte tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß), and enhanced the expression of nutrient factors, including brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1). The Notch signaling pathway specific inhibitor, N-[N-(3,5-Difluorophenacetyl)-1-alany1]-S-phenyglycine t-butylester (DAPT), could significantly enhance the effect of GAS on TNC1 astrocytes after OGD, such as the inhibition of inflammatory factors and the up-regulation of neurotrophic factors. CONCLUSIONS: GAS exerts dual effects on astrocytes via regulation of the Notch signaling pathway. We found that it could inhibit the pro-inflammatory factors mediated by astrocytes, and also promote the secretion of neurotrophic factors by astrocytes. These results provide a new biological mechanism for the treatment of neuroinflammatory diseases by GAS.

Gastrodin Regulates the Notch Signaling Pathway and Sirt3 in Activated Microglia in Cerebral Hypoxic-Ischemia Neonatal Rats and in Activated BV-2 Microglia.

In response to hypoxic-ischemic brain damage (HIBD), microglia activation and its mediated inflammation contribute to neuronal damage. Inhibition of over-activated microglia is deemed to be a potential therapeutic strategy. Our previous studies showed that gastrodin efficiently depressed the neuroinflammation mediated by activated microglia in HIBD neonatal rats. The underlying mechanisms through which gastrodin acts on activated microglia have not been fully elucidated. This study is designed to determine whether gastrodin would regulate the Notch signaling pathway and Sirtuin3 (Sirt3), which are implicated in regulating microglia activation. The present results showed that gastrodin markedly suppressed the expression of members of Notch signaling pathway (Notch-1, NICD, RBP-JK and Hes-1) in activated microglia both in vivo and in vitro. Conversely, Sirt3 expression was enhanced. In BV-2 microglia treated with a γ-secretase inhibitor of Notch pathway- DAPT, the expression of RBP-JK, Hes-1, and NICD was suppressed in activated microglia. Treatment with DAPT and gastrodin further decreased NICD and Hes-1 expression. Sirt3 expression was also decreased after DAPT treatment. However, Sirt3 expression in activated BV-2 microglia given a combined DAPT and gastrodin treatment was not further increased. In addition, combination of DAPT and Gastrodin cumulatively decreased tumor necrosis factor-α (TNF-α) expression. The results suggest that gastrodin regulates microglia activation via the Notch signaling pathway and Sirt3. More importantly, interference of the Notch signaling pathway inhibited Sirt3 expression, indicating that Sirt3 is a downstream gene of the Notch signaling pathway. It is suggested that Notch and Sirt3 synergistically regulate microglia activation such as in TNF-α production.

Scutellarin Exerts Anti-Inflammatory Effects in Activated Microglia/Brain Macrophage in Cerebral Ischemia and in Activated BV-2 Microglia Through Regulation of MAPKs Signaling Pathway.

BACKGROUND: Scutellarin, an herbal compound, can effectively suppress the inflammatory response in activated microglia/brain macrophage(AM/BM) in experimentally induced cerebral ischemia; however, the underlying mechanism for this has not been fully clarified. We sought to elucidate if scutellarin would exert its anti-inflammatory effects on AM/BM through the MAPKs pathway. MATERIALS AND METHODS: Western blot and immunofluorescence labeling were used to determine the expression of the MAPKs pathway in AM/BM in rats subjected to middle cerebral artery occlusion (MCAO) also in lipopolysaccharide (LPS)-activated BV-2 microglia in vitro. Furthermore, expression of p-p38 along with that of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta(IL-1ß), and inducible nitric oxide synthase (iNOS) in LPS-activated microglia subjected to pretreatment with p38 inhibitor SB203580, p38 activator sc-201214, scutellarin, or a combination of them was evaluated. FINDINGS: Scutellarin markedly attenuated the expression of p-p38, p-JNK in AM/BM in MCAO rats and in vitro. Conversely, p-ERK1/2 expression level was significantly increased by scutellarin. Meanwhile, scutellarin suppressed the expression of proinflammatory mediators including iNOS, TNF-α, and IL-1ß in AM/BM. More importantly, SB203580 suppressed p-p38 protein expression level in LPS-activated BV-2 microglia that was coupled with decreased expression of proinflammatory mediators (TNF-α, iNOS) in LPS-activated BV-2 microglia. However, p38 activator sc-201214 increased expression of proinflammatory mediators TNF-α, iNOS, and IL-1ß. Interestingly, the decreased expression of both proinflammatory markers by p38 MAPK inhibitor and increased expression of proinflammatory markers by p38 MAPK activator were compatible with that in BV-2-activated microglia pretreated with scutellarin. CONCLUSIONS: The results suggest that scutellarin down-regulates the expression of proinflammatory mediators in AM/BM through suppressing the p-JNK and p-p38 MAPKs. Of note, the anti-inflammatory effect of p38 MAPK inhibitor and scutellarin is comparable. Besides, p38 MAPKs activator reverses the effect of scutellarin. Additionally, scutellarin increases p-ERK1/2 expression that may be neuroprotective.

[Risk factors of pinhole infection in the fractured lower limbs after external fixation: a case-control study].

OBJECTIVE: To investigate the risk factors of pinhole infection in the fractured lower limbs after external fixation. METHODS: The case-control study was designed. From May 2009 to May 2014, the clinical data of 272 patients with lower limb fracture treated by external fixation device were collected. All the patients were divided into two groups according to post-operative pinhole infection. There were 29 cases in the case group including 23 males and 6 females. The age of patients in case group ranged from 25 to 77 years old,with the average age of (53.41 ± 12.77) years old. There were 243 cases in control group including 217 males and 26 females. The age of patients in the control group ranged from 27 to 78 years old, with the average age of (48.71 ± 11.87) years old. There were nine risk factors observed in our study including age, gender, fixed time by external fixation device, diabetes, time in bed, smoking, operation condition of other parts in the body, infection condition of other parts in the body. RESULTS: The results of univariate analysis showed that there were statistically significant differences among age (χ² = 15.708, P < 0.001), fixed time by external fixation device (χ² = 11.940, P < 0.001), severity of the lower limb fracture (χ² =15.438, P < 0.001), diabetes (χ² = 8.519, P = 0.004) and time in bed (χ² = 7.165, P = 0.007) between case group and control group. The results of Logistic regression analysis showed that the risk factors of pinhole infection after fixed by external fixation device in the lower limb fracture were the advanced age (OR = 8.327, P < 0.001), fixed time by external fixation device (OR = 6.795, P < 0.001), diabetes (OR = 4.965, P = 0.001) and time in bed (OR = 4.864, P = 0.008). CONCLUSION: The advanced age, long fixed time, diabetes and long time in bed could increase the risk of pinhole infection after external fixation in the lower limbs with fracture.

Expression of angiotensin II and its receptors in activated microglia in experimentally induced cerebral ischemia in the adult rats.

Expression of angiotensin II (Ang II) and its receptors (AT1/AT2) is undetected in the mature microglia in normal brain. We report here that the immunoexpression of Ang II and AT1/AT2 was altered in activated microglia notably at 1 week in rats subjected to middle cerebral artery occlusion (MCAO). Immunolabeled activated microglia were widely distributed in the infarcted cerebral tissue after MCAO. By enzyme immunoassay, Ang II protein expression levels of the ischemic tissues were decreased drastically at 12 h after ischemia, then rose rapidly at 3 days and 1 week after MCAO when compared with the control. On the other hand, AT1 and AT2 receptor mRNA and protein levels were up-regulated after MCAO, peaking at 12 h, but declined thereafter. Expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) mRNA and protein levels was concomitantly increased. Edaravone significantly suppressed Ang II and AT1/AT2 receptor expression as well as that of TNF-α and IL-1ß suggesting that microglia-derived Ang II can act through an autocrine manner via its receptor that may be linked partly to the production of proinflammatory cytokines. We conclude that neuroinflammation in MCAO may be attenuated by Edaravone which acts through suppression of expression of Ang II and its receptors and proinflammatory cytokines in activated microglia.

Expression of Kv1.2 in microglia and its putative roles in modulating production of proinflammatory cytokines and reactive oxygen species.

Microglial cells are endowed with different potassium ion channels but their expression and specific functions have remained to be fully clarified. This study has shown Kv1.2 expression in the amoeboid microglia in the rat brain between 1 (P1) and 10 (P10) days of age. Kv1.2 expression was localized in the ramified microglia at P14 and was hardly detected at P21. In postnatal rats exposed to hypoxia, Kv1.2 immunoreactivity in microglia was markedly enhanced. Quantitative RT-PCR analysis confirmed Kv1.2 mRNA expression in microglial cells in vitro. It was further shown that Kv1.2 and protein expression coupled with that of interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) was significantly increased when the cells were subjected to hypoxia. The same increase was observed in cells exposed to adenosine 5'-triphosphate (ATP) and lipopolysaccharide (LPS). Concomitantly, the intracellular potassium concentration decreased significantly. Blockade of Kv1.2 channel with rTityustoxin-Kalpha (TsTx) resulted in partial recovery of intracellular potassium concentration accompanied by a reduced expression of IL-1beta and TNF-alpha mRNA and protein expression and intracellular reactive oxygen species (ROS) production. We conclude that Kv1.2 in microglia modulates IL-1beta and TNF-alpha expression and ROS production probably by regulating the intracellular potassium concentration.

[Ultrastructural dynamic observation on murine schistosomal hepatic fibrosis].

OBJECTIVE: To explore possible mechanisms of hepatic fibrosis by investigating the ultrastructural dynamic changes of liver tissue, especially several kinds of cells related to hepatic fibrosis. METHODS: Murine schistosomal hepatic fibrosis model was established by infecting mice with Schistosoma japonicum cercariae. Routine transmission electron microscopy was used to observe the liver tissue. H.E. staining was used for examining the pathological changes. RESULTS: H.E. staining showed that the model was established successfully. Ultrastructural observation showed that at the 6th week after infection, the necrosis of hepatocytes around the acute granulomas occurred; the number of sinusoidal endothelial fenestrae and vitamin A droplets in fat-storing cells decreased; large phagosomes and rough endoplasmic reticulum could be seen in the cytoplasm of Kupffer's cells. At the 8th week, steatosis was found in some hepatocytes, some microvilli emerged on a few inter-hepatocytic surfaces and the inter-hepatocytic spaces were enlarged. Large collagen fibrillar bundles filled in the perisinusoidal spaces, and capillarization of hepatic sinusoids was observed. Secretory vesicles filled with collagen fibrils appeared in the cytoplasm of fat-storing cells with large amount of collagenous fiber bundles surround the cells. Rough endoplasmic reticulum increased in Kupffer's cells. At the 10th week, fat-storing cells were activated and transformed into myofibroblasts. At the 12th week, the number of myofibroblasts decreased but that of fibroblasts and fiber cells increased. CONCLUSION: Activation of fat-storing cells and transformation from fat-storing cells into myofibroblasts are the critical link in the development of hepatic fibrogenesis following schistosome infection. Kupffer's cells, necrotic hepatocytes and sinusoidal endothelial cells may relate to the activation of fat-storing cells. Capillarization of hepatic sinusoids possibly accelerates the development of hepatic fibrosis.