Astrocytes from MHE rats overexpress S100B
In astrocytes, S100B, a cytokine marker of astroglial activation in brain disorders, induces pro-inflammatory mediators' secretion . Thus, we sought to determine whether S100B was elevated in astrocytes in neurological diseases MHE. We examined the expression of S100B using IB analysis and found that the expression of S100B was significantly increased in cortexes of MHE rats (Figure 1A and 1B). A maximal increase in S100B mRNA transcription was observed in cortexes (qPCR, Figure 1C and 1D), suggesting overexpression of S100B in the MHE brain.
To further investigate astrocytes' role in cortexes of MHE rats, we established adult astrocytes culture from MHE rats and WT rats. The astrocyte cell culture purity was analyzed by immunofluorescence staining with an astrocyte-specific marker (GFAP) and other cell-type-specific markers for neurons (MAP2), microglia (IBA1), endothelial cells (CD31), and oligodendrocytes (O4). We found that 99% of the cells were stained with GFAP (Figure 1E). We further evaluated our cell culture using RT-PCR by targeting these specific cell markers. We found that cultured astrocytes had an enriched expression of astrocyte-specific gene GFAP and low expression of other cell-type-specific genes compared to the whole brain (Figure 1F). Altogether, these experiments showed that this culture protocol generates a highly enriched astrocyte culture. Then, S100B production and secretion were assessed using IB analysis, qPCR, and ELISA assay. IB analysis and qPCR assay showed that MHE astrocytes showed a significantly higher S100B protein (Figure 1G and 1H) and mRNA expression (Figure 1I and 1J) compared to WT astrocytes. Moreover, an ELISA assay suggested that the level of S100B released from MHE astrocytes into the cell culture medium was obviously elevated compared to WT astrocytes (Figure 1K). Thus, we deduced there was an overload of S100B in MHE astrocytes.
Furthermore, to demonstrate the specificity of S100B in inflammation and oxidative stress, we inhibited S100B using an anti-S100B antibody in media and siRNA-induced-silencing in MHE astrocytes. We first tested the efficiency of S100B siRNA transfection into MHE astrocytes. As determined by qPCR, MHE astrocytes showed weak S100B expression after S100B siRNA transfection (Figure 1L) compared to control siRNA transfection, indicating efficient transfection. Then, we measured the inflammatory factors expression and secretion in MHE astrocytes. We found that the level of TNFα in the cell culture medium of MHE astrocytes was significantly increased compared to WT astrocytes. After knocking down the S100B in MHE astrocytes, the TNFα secretion was reduced or even inhibited (Figure 1M), indicating the induction of inflammation in MHE astrocytes by S100B.
MHE astrocytes are associated with S100B and VEGF production
Astrocyte-derived VEGF, as a pro-inflammatory cytokine , has a significant role in lesion pathogenesis of CNS disease characterized by inflammation [17, 33]. In this study, we investigated whether autocrine pro-inflammatory S100B from astrocytes affects the VEGF production and secretion. First of all, we examined the association of S100B and VEGF in MHE astrocytes in vivo and in vitro. Immunostaining of the MHE rat's brain revealed that S100B was colocalized with VEGFa in astrocytes (Figure 2A). Furthermore, confocal images from MHE astrocytes cultured in vitro showed colocalization of S100B with VEGF-A in the cytoplasm (Figure 2B).
Next, we addressed whether MHE astrocytes-derived S100B enhances VEGF production in an autocrine manner using S100B siRNA transfected-MHE astrocytes. IB and qPCR analysis showed significant elevation in VEGF protein (Figure 2C and 2D) and mRNA expressions (Figure 2E and 2F) in MHE astrocytes; however, these levels were decreased in cells transfected with S100B siRNA. Similar results were obtained from the ELISA assay; the cell culture medium of MHE astrocytes transfected with the control vector showed a significant increase in VEGF level, while S100B-deficient MHE astrocytes displayed decreased VEGF secretion (Figure 2G). Hence, our data suggested that autocrine S100B facilitated VEGF overproduction by MHE astrocytes.
Next, we assessed whether VEGF autocrine accumulation impacted the S100B expression in MHE astrocytes using VEGF siRNA transfection into MHE astrocytes. VEGF siRNA obviously decreased VEGF mRNA in MHE astrocytes by qPCR assay (Figure 2H), indicating successful transfection of VEGF siRNA. More importantly, IB analysis for S100B expression showed no change in S100B protein level after VEGF siRNA transfection in MHE astrocytes (Figure 2I and 2J). Additionally, the ELISA assay also showed that the level of S100B in the cell culture medium of MHE astrocytes was not altered after AVAB treatment (Figure 2K), indicating that changes in VEGF do not affect the S100B overload.
S100B-enhances VEGF production through a TLR2 and RAGE interaction
TLRs are involved in the release of inflammatory cytokines . A previous study suggested that S100B protein binds to RAGE and, in turn, activates TLRs . We estimated that autocrine S100B might trigger TLR activation via stimulation of RAGE, leading to VEGF production in MHE astrocytes. Thus, we tested the binding between RAGE and S100B in vitro using a coimmunoprecipitation experiment. The results showed that RAGE coimmunoprecipitated with S100B in MHE astrocytes (Figure 3A). We also validated this result by performing a reciprocal experiment using anti-rage, and found that RAGE and S100B physically interacted in MHE astrocytes (Figure 3A)
Next, we analyzed the association of RAGE with TLR in MHE rats in vivo using immunostaining experiments. Confocal images showed the co-localization of TLR2 with RAGE combined with GFAP in astrocytes in cortexes in MHE rats (Figure 3B), whereas the co-localization of TLR4 with RAGE was not found in astrocytes of cortexes (Figure 3C).
Furthermore, we tested the effect of S100B on the interaction between RAGE and TLR2 or TLR4 in MHE astrocytes in vitro using co-immunoprecipitation experiments. The results showed that RAGE was coimmunoprecipitated with TLR2 but no TLR4 in MHE astrocytes. We also validated this result by performing a reciprocal experiment using anti-TLR2. RAGE and TLR2 physically interacted, while RAGE failed to be coimmunoprecipitated with TLR2 after ASAB treatment (Figure 3D). Thus, we deduced that S100B elicits the RAGE and TLR2 but no RAGE and TLR4 interaction.
To assess whether S100B stimulates the VEGF expression through RAGE/TLR2 signaling, we incubated MHE astrocytes with RAGE inhibitor FPS-ZM1 and TLR2 inhibitor C29 preincubated with various concentration of S100B. Quantitative analysis by IB showed an increase in S100B-dependent VEGF production in a dose-response manner. To evaluate the contribution of RAGE and TLR2 interaction to S100B activity, we used RAGE inhibitor FPS-ZM1 and TLR2 inhibitor C29 in a competition assay. We discovered that FPS-ZM1 or C29 significantly reduced the effect of a high dose of S100B on VEGF expression in MHE astrocytes (Figure 3E and 3F). Similar results were found by ELISA assay; FPS-ZM1 or C29 dramatically decreased the high dose of S100B-induced VEGF secretion in MHE astrocytes (Figure 3G). Hence, we deduced that RAGE and TLR2 are required for S100B-mediated VEGF production.
Autocrine S100B-mediated VEGF triggers VEGFR2 and COX-2 interaction
The roles of VEGF receptors  and COX-2  are associated with inflammatory processes. VEGFR2 participates in VEGF-mediated COX induction . Therefore, we categorized the contribution of VEGFR2 or COX-2 to S100B-mediated VEGF activity. First, we used mouse anti-COX-2 and anti-GFAP antibodies combined with VEGFR1/2 to test the co-localization of VEGFR1/2 and COX-2 in vivo. Confocal images confirmed the presence of VEGFR2, but not VEGFR1, colocalized with COX-2 in the cortex astrocytes of MHE rats (Figure 4A and 4B). Next, we assessed the effect of S100B on the interaction between VEGFR and COX-2 in MHE astrocytes in vitro using co-immunoprecipitation analysis. As shown in Figure 4C, substantial COX-2 was coimmunoprecipitated with abundant VEGFR2 but not with VEGFR1. We also validated this result by performing a reciprocal experiment using anti-VEGFR2. Our result indicated that VEGFR2 and COX2 physically interacted in MHE astrocytes, whereas ASAB addition induced no interaction of VEGFR2 with COX2, thus suggesting that S100B facilitates the interaction of VEGFR2 with COX-2.
Next, to investigate whether VEGFR2/COX-2 signaling was implicated in S100B-mediated autocrine VEGF, we treated MHE astrocytes with AVAB or VEGF siRNA transfection in the presence of S100B. qPCR analysis showed that AVAB decreased the VEGFR2 mRNA expression in MHE astrocytes (Figure 4D and 4E). Moreover, the knockdown of VEGF and AVAB attenuated the effect of S100B and decreased the expression of COX-2 (Figure 4F-H). Hence, our data indicated that VEGF is required for S100B-mediated VEGFR2/COX-2 signaling.
S100B-mediated activation of NFƙB depends on autocrine VEGF by astrocyte
Nuclear factor kappa-light-chain-enhancer of activated B cells (NFƙB), as a central transcription factor, has a major role in pro-inflammatory response. Studies have found that S100B can induce nuclear translocation and transcriptional activity of NFƙB (p65) . However, the exact mechanism of S100B-mediated NFƙB activation in an autocrine action still remained unclear. Thus, we examined the contribution of S100B autocrine to activation of NFƙB in a competition assay. Immunoblots of cytoplasmic and nuclear NFƙB protein showed that nuclear NFƙB content was significantly increased in MHE astrocytes. Moreover, the addition of S100B substantially enhanced NFƙB nuclear translocation. Then, we evaluated the contribution of VEGF to S100B activity. Silencing of VEGF by VEGF siRNA transfection significantly diminished S100B-mediated NFƙB nuclear translocation in MHE astrocytes, indicating the involvement of VEGF in the S100B-response NFƙB nuclear translocation (Figure 5A-C).
Next, we used a luciferase-based reporter gene construct to test an NFƙB-sensitive gene transcription. NFƙB-dependent transcription indeed was activated in MHE astrocytes, and the enhancement of transcriptional response was observed after treating cells with exogenous S100B. However, knockdown of VEGF effectively blocks the S100B-mediated activation of the NFƙB-dependent transcription (Figure 5D), indicating the role of VEGF in the S100B-response NFƙB activation.
To assess whether S100B affects NFƙB activation through COX-2, we treated MHE astrocytes with S100B together with VEGFR2 inhibitor PTK787 or COX-2 inhibitor celecoxib. Immunostaining confirmed that NFƙB was largely localized to the nucleus in S100B-treated MHE astrocytes. After AVAB, PTK787, or celecoxib treatment, NFƙB was hardly detected in the cell nucleus (Figure 5E), suggesting an involvement of VEGFR2 and COX-2 in S100B-dependent NFƙB nuclear translocation. We then examined the levels of pro-inflammatory factors using qPCR analysis. S100B induced an excessive TNFα level in MHE astrocytes, while AVAB addition significantly diminished S100B-induced TNFα production (Figure 5F and 5G), indicating the responsibility of VEGF for the S100B-induced inflammatory action.
S100B-mediated oxidative stress depends on autocrine VEGF by astrocytes
Elevated oxidative stress  and inflammation,  which have been associated with the severity of disease, have been reported in a variety of pathophysiological conditions. The linkage between increased oxidative stress and acute-phase inflammation is already well understood . Like inflammation, S100B is implicated in oxidative stress. In this study, we transfected MHE astrocytes with S100B siRNA to determine the contribution of S100B in oxidative stress. ROS production assay showed that ROS level was obviously increased in MHE astrocytes. However, the ROS production was significantly lowered with the knockdown of S100B (Figure 6A). Moreover, IB analysis of NADPH oxidase expression showed significant increases in NOX1/NOX2 level in MHE astrocytes, while the knockdown of S100B significantly reduced the protein expression (Figure 6B and 6C). In addition, analysis of NADPH oxidase activity displayed an obvious increase in MHE astrocytes, while ASAB treatment led to decreased activity (Figure 6D), indicating the induction of inflammation in MHE astrocytes by S100B.
The binding of VEGF to VEGFR2 results in a burst of ROS . To examine whether S100B autocrine enhances the oxidative stress through VEGF autocrine in MHE astrocytes, we pre-incubated MHE astrocytes with S100B and then transfected cells with VEGF siRNA. The addition of S100B further amplified ROS production, while the silencing of VEGF lowered the levels of ROS in S100B-treated MHE astrocytes, indicating the responsibility of VEGF for the S100B-induced inflammatory action (Figure 7A). Moreover, the knockdown of VEGF significantly diminished the action of S100B, indicating the involvement of VEGF in the S100B-response NOX1 and NOX2 regulation (Figure 7B and 7C).
NF-kB up-regulates NADPH oxidase activity, leading to an increase in ROS levels . COX2 contributes to oxidative stress  and regulates NADPHox-driven ROS production . To assess the impact of S100B on the oxidative stress depending on COX-2 and NFƙB, we treated MHE astrocytes with S100B together with NFKB inhibitor PDTC or COX-2 inhibitor celeCOXib. NADPH oxidase activity was significantly increased in MHE astrocytes; increased NADPH oxidase activity was further activated after treatment of exogenous S100B. However, PTK787, celeCOXib, PDTC, or AVAB addition diminished the effect of S100B (Figure 7D), indicating the involvement of COX-2/nfkb signaling in S100B-response oxidative stress.
MHE astrocytes have impaired ability to promote neuronal growth
Given the extensive dependency of neurons on astrocytic support (growth and viability), we co-cultured N2A cells with MHE astrocytes to address the effect of the MHE astrocytes on the neuronal viability using MTT assay. MHE astrocytes resulted in obvious N2A cell loss after 18h and 24h of co-culturing (Figure 8A). Moreover, we evaluated the apoptotic effect of MHE astrocytes on N2A cells in vitro by flow cytometry. Flow cytometric analysis of apoptosis showed that the percentage of apoptotic neurons was significantly higher after co-culturing N2A cells with MHE astrocytes than with WT astrocytes (Figure 8B and 8C). Moreover, similar results were observed in vivo. Briefly, we isolated neurons from P1–P2 pups with co-cultured of MHE astrocytes to assess primary cortical neuronal apoptosis using the TUNEL study. Primary cortical neurons showed the obvious increase in TUNEL positive cells (apoptosis) in co-cultures with MHE astrocytes as compared to co-cultures with MHE astrocytes (Figure 8D).
We also characterized whether MHE astrocytes were impaired in promoting neurite growth length of the longest neurite per primary cortical neuron. Length of the longest neurite per neuron, evident by β-tubulin staining, was measured in at least 100 neurons per coverslip. We discovered that neurons had longer neurites in contact with WT astrocytes. MHE astrocytes promoted the growth of shorter neurites by co-cultures as compared to WT astrocytes culture (Figure 8E and 8F). These data indicated that activated astrocytes impair neuronal survival in MHE.