Gastrodin regulates expression of angiotensin-SirT3 and proinflammatory mediators in reactive astrocytes via activated microglia

doi:10.21203/rs.3.rs-2122250/v1

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Gastrodin regulates expression of angiotensin-SirT3 and proinflammatory mediators in reactive astrocytes via activated microglia

https://doi.org/10.21203/rs.3.rs-2122250/v1

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Use of Chinese herbal compounds have been widely explored in recent years to inhibit excessive proliferation and activation of astrocytes in brain injuries. Thus, it has been reported that Gastrodin, an anti-inflammatory agent, can effectively suppress microglia activation in rats with hypoxic-ischemia brain damage. This study was therefore aimed to determine if Gastrodin would exert a similar effect on reactive astrocytes and alleviate their inflammatory response through the renin-angiotensin system (RAS) and Sirtuin3 (SirT3). Expression of ATO, AT₁ and SirT3 was detected in TNC-1 astrocytes, treated respectively with conditioned medium from BV-2 microglia with or without pretreatment of Gastrodin and lipopolysaccharide (LPS) by immunofluorescence and Western blotting. Along with this, expression of C3, S100A10, TNF-α and neurotrophic factors, including BDNF and IGF-1 expression was also evaluated. We showed here that TNC-1 astrocytes incubated in CM + LPS showed a marked increase in ATO, AT₁, C3 and TNF-α when compared to cells in the BV-2 conditioned medium (CM), but that of S100A10 was reduced. Of note, TNC-1 astrocytes responded vigorously to conditioned medium derived from BV-2 microglia treated with Gastrodin + LPS when compared to the controls. This was evident by the decreased expression of the above-mentioned proteins, except for S100A10. As opposed to this, SirT3, IGF-1 and BDNF expression as determined by Western and immunofluorescence analysis was enhanced. The results suggest that Gastrodin inhibits the expression of RAS and proinflammatory mediators, but promotes the production of large amounts of neurotrophic factors in reactive astrocytes induced by LPS-stimulated activated microglia. Another novel finding of this study is the unequivocal demonstration that Gastrodin can promote the polarization of A2 phenotype that is regarded to be neuroprotective. This has added a new therapeutic perspective of this herbal compound that is gaining more attention in recent years.

astrocytes

angiotensin-sirt3

Gastrodin

microglia

Gastrodin (C13H18O7) is a phenolic glycoside compound and the main bioactive component of Gastrodia elata^[1]. It increases blood supply, relieves pain, and anticonvulsants^[2]. Furthermore, Gastrodin exerts neuroprotective effects in different neurological diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and cerebral ischemia^[3–5]. Studies have reported that Gastrodin can inhibit the expression of NO, COX-2 and pro-inflammatory cytokines in LPS-stimulated microglia via the MAPK pathway^[6]. In stroke, Gastrodin attenuates brain injury by enhancing antioxidant and anti-inflammatory activities and inhibiting apoptotic pathways^[7]. We reported recently that Gastrodin effectively suppressed production of proinflammatory mediators in activated microglia in cerebral hypoxic-ischemia neonatal rats and in activated BV-2 microglia^{[8, 9]}; hence, understanding the roles of microglia activation is crucial to develop the therapeutic potential of Gastrodin for stroke.

Microglia activation is a major cause of inflammation in the central nervous system (CNS), providing the first line of defense in the event of injury or disease^{[10, 11]}. It plays a pivotal role in the inflammatory response in the CNS^[12]. Activated microglia secrete a multitude of pro-inflammatory mediators and induce astrocytes to produce different secondary inflammatory molecules^[13]. Astrocytes play an important role in brain homeostasis^[14] and participate in local innate and adaptive immune responses in the CNS^[15]. They help regulate blood flow, maintain the blood-brain barrier (BBB), supply neurons with energy substances, modulate synaptic activity, control neurotrophic hormone secretion, clear dead cells, regulate extracellular ion, fluid and transmitter balance, and scarring^{[14, 16, 17]}. Many recent studies have reported that reactive astrocytes present two distinct phenotypes: namely, A1 and A2 astrocyte. A1 astrocytes exert neurotoxic effects, whereas A2 astrocytes are neuroprotective^[18]. In the latter study, it has been found that activated microglia can induce astrocyte to A1 phenotype by releasing interleukin-1α (IL-1α), tumor necrosis factor (TNF-α), and complement component 1q (C1q)^[18]. In traumatic brain injury, microglia can induce astrocyte transition to a neuroprotective phenotype by downregulating P2Y1 receptors^[19].

The renin-angiotensin system (RAS) has important physiological functions in regulating water and electrolyte balance, systemic vascular resistance, blood pressure, and cardiovascular homeostasis^[20]. The system comprises different components including angiotensinogen (ATO), angiotensin peptide and its cognate receptors^[21]. Among them, angiotensin II (Ang II) affects cell survival, differentiation and inflammation in addition to vascular and renal effects^[22–24]. Studies have demonstrated the localization of RAS in the substantia nigra of rodent and primate brains that modulates dopamine release and dopamine receptor expression^[25]. Interestingly, long-term blockade of brain Ang II-AT₁ receptors can ameliorate cerebral ischemia injury. Meanwhile, it is noteworthy that anti-apoptotic mechanisms and suppression of post-ischemic inflammation were associated with AT₁ receptor blockade-induced neuroprotection in ischemic brain tissue^[26]. In light of the above, this study was conducted to explore whether Gastrodin would regulate the inflammatory responses by elicited by RAS in reactive astrocytes through activated microglia.

Relevent to the above is Sirt3, which has been reported to protect against AngII-induced cellular dysfunction by regulating ROS production^[27]. Sirtuin 3 (Sirt3), a member of the Sirtuins protein family, is a reactive deacetylase that regulates metabolism and oxidative stress^[28]. Sirt3 can ameliorate brain injury by mediating mitochondrial ceramide synthesis^[29]. Previous studies have demonstrated that Gastrodin can inhibit microglial activation through suppression of the Notch-1 and the Sirt3 pathways^[9].

The present study was conducted to determine the expression changes and roles of RAS and SirT3 in astrocyte reaction mediated by activated microglia in vitro. We have reported previously that expression of proinflammatory mediators and RAS in activated microglia was significantly suppressed by Gastrodin. We have extended this in the present study to ascertain if these biomarkers were expressed in reactive astrocytes, and, if so, whether it would also be affected by Gastrodin. Furthermore, we sought to determine if Gastrodin would exert its beneficial effect by regulating the production of proinflammatory mediators in both astrocyte phenotypes and whether this might be through the ATO/AT₁-SirT3 pathways.

2.1 BV-2 microglia and TNC-1 astrocyte cultures

Both BV-2 microglial cells and TNC-1 astrocytes were respectively cultured in DMEM supplemented with 10% FBS at 37°C in a humidified 5% CO₂ incubator. According to the cell count results, BV-2 microglial cells were seeded 6 x 10⁵ cells per well in a 6-well plate; TNC-1 astrocytes were seeded 3 x 10⁶ cells per well. Lipopolysaccharide (LPS) or lipopolysaccharide induced BV-2 microglia conditioned medium was used to co-activate or act separately TNC-1 astrocytes along with the control group. The proteins of TNC-1 astrocytes subjected to different treatments were extracted after incubation at 37°C in a 5% CO₂ incubator for 24 hours. TNC-1 astrocytes divided from different interventions were also processed for immunofluorescence study.

2.2 Cell viability assay of BV-2 microglia and TNC-1 astrocytes

To determine the cytotoxic effect of Gastrodin to BV-2 microglia and Azilsartan to TNC-1 astrocytes, BV-2 microglia and TNC-1 astrocytes were, respectively, plated into 96-well microplates (10³ cells/well) and cultured for 24h. They were subjected to treatment of Gastrodin (0 to 1mg/ml) and Azilsartan (0 to 160µM) in each well containing 200 µl of culture medium for 1h. The cells were taken out and incubated in cell counting kit-8 (CCK-8) solution under dark conditions (CCK-8 solution: medium = 1:100), and then incubated in an incubator at 37 ℃ for 3 h. Finally, the corresponding absorbance [optical density (OD)] value of the cells was read at a wavelength of 450 nm using the microplate analyzer. This test was repeated three times, and the average value was taken for statistical analysis. The assays were performed in triplicate.

2.3 Preparation and use of BV‑2 conditioned medium

At 24 h after BV-2 cell seeding, the medium was discarded. Four different kinds of conditioned medium were prepared: BV-2 conditioned medium as control (CM): incubation of BV-2 cells was in 1 mL basic DMEM for 3 h; BV-2 conditioned medium + Lipopolycharride (LPS) (CM + LPS): BV-2 cells were incubated with 1 mL basic medium with 1 µg/ml of LPS for 3 h; BV-2 conditioned medium with Gastrodin pretreatment + LPS (CM + LPS + G_L and CM + LPS + G_H): for this, BV-2 cells were first incubated with 1 ml basic medium containing 0.17mM and 0.34 mM of Gastrodin for 1 h. The medium was then discarded and the cells were washed with PBS twice. Following this, 1 ml basic medium containing 1 µg/ml of LPS was added for another 3 h. All 4 conditioned media were collected and filtered through 0.22 µm syringe filters. The media prepared were then ready to use for treatment of TNC-1 astrocytes.

2.4 Treatment of TNC-1 astrocytes with Azilsartan

TNC-1 astrocytes were seeded in six-well plates with a density of 3 × 10⁶ cells/mL per well. The cells were pretreated with Azilsartan (APExBIO) (50 µM) which was dissolved in DMSO, as recommended by the manufacturer’s instructions for 1 h before other treatments in a 37°C, 5% CO₂ humidified incubator. Next, under the same conditions, the cells were treated 24h with BV‑2 conditioned medium.

2.5 TNC1 astrocytes and treatment with BV‑2 conditioned medium

TNC1 astrocytes were divided into control, direct stimulation with LPS (LPS), BV-2 conditioned medium (CM) as control; CM + Lipopolycharride (CM + LPS); CM + Lipopolycharride + Gastrodin (CM + LPS + G_L, CM + LPS + G_H); Azilsartan treated (CM + A); CM + Lipopolycharride + Azilsartan (CM + LPS + A); CM + Lipopolycharride + Azilsartan + Gastrodin (CM + LPS + A + G_L) groups based on treatment with the respective conditioned medium. TNC1 cells were seeded at a density of 3×10⁶ in 6-well plates and 5 × 10⁴ in 24 well plates, incubated with a complete medium. After 24 h, the medium was discarded and the cells were incubated with 1 ml (6-well plates) or 500 µl (24-well plates) of conditioned medium for 24 h. The cells were then harvested for protein extraction and immunofluorescence microscopy.

2.6 Western blot analysis of TNC-1 astrocytes

After incubating with different media for 24 h, TNC1 astrocytes of different groups were treated with lysis buffer after rinsing twice with PBS. Sixty µl of lysate was added to each well and the mixture well shaken well. The scraped cells in the well were left at 4 ° C for 10 min, and shaken 3 times during this period. The lysate was centrifuged at 14,000 rpm and 4 ° C for 20 min and the supernatant collected for further analysis. The concentration of the extracted protein was then determined using a BCA kit. Equivalent amounts of protein were prepared for 10% sodium lauryl sulfate-polyacrylamide electrophoresis. Electrophoretic separation was performed and the separated proteins transferred to a polyvinylidene fluoride membrane. Blocking was with skim milk for 1-2h.The membranes were incubated with β-actin (Mouse monoclonal IgG 1:5000; Sigma;), C3(rabbit monoclonal IgG 1:1000; abcam), S100A10 (mouse monoclonal IgG 1:1000;Thermo Fisher), AT₁ (rabbit polyclonal IgG 1:500; abcam), ATO (rabbit polyclonal IgG 1:1000; ORIGENE), TNF-α (rabbit polyclonal IgG 1:1000; Milipore), SirT3 (rabbit polyclonal IgG 1:500; Santa Cruz Biotechnology), IGF-1(rabbit polyclonal antibody 1:1000;ORIGENE ), BDNF(rabbit polyclonal antibody 1:1000༛ORIGENE) primary antibodies diluted in blocking solution overnight at 4 ℃. Following incubation, the membranes were removed and rinsed quickly three times; secondary antibodies (Goat anti-Mouse IgG, Peroxidase Conjugated; 1: 5000; Milipore; USA, Goat anti-Rabbit IgG, Peroxidase Conjugated; 1: 5000; Milipore; USA) were then added and incubated at room temperature 1h. The band was then detected with a chemiluminescence imager.

2.7 Immunofluorescence labeling of TNC-1 astrocytes

The cell slices were placed in a 24-well plate. Diluted polylysine was then added to each well. TNC-1 astrocytes were grouped and processed according to the cell culture steps described above. The treated cells were washed twice with 0.01M PBS, and then a 4% paraformaldehyde solution was added to each well and fixed for 20 min. The cells were washed twice with PBS and 5% goat serum was added to each well. After this, they were incubated at room temperature for 1h. Primary antibody ATO (rabbit polyclonal IgG 1:100; ORIGENE), AT₁ (rabbit polyclonal IgG 1:100; ORIGENE), SirT3 (Mouse monoclonal antibody 1:50; Santa Cruz Biotechnology), IGF-1(rabbit polyclonal antibody 1:200;ORIGENE), BDNF (rabbit polyclonal antibody 1:100; ORIGENE) and GFAP (Mouse、rabbit monoclonal antibody 1:100; ORIGENE) was then added to each well and incubated at 4 ℃ overnight. Incubated cells were rinsed three times with PBS, followed by incubation with fluorescent secondary antibodies: Cy3 labeled goat anti-rabbit IgG (1: 200; Sigma) and FITC labeled goat anti-mouse IgG (1: 200; Sigma) to each well at room temperature for 1 h. Following rinsing three times with PBS, the cell slices were removed. A small amount of fluorescent mounting was medium was added. Images were captured with a fluorescence microscope.

2.8 Statistical analysis

Statistical analysis was performed by SPSS 17.0 statistical software. The measurement data were tested for normality and homogeneity of variance. The data of each group have been expressed as mean ± standard error. After homogeneity test of variances, one-way Analysis of Variance (ANOVA) followed by multiple comparison of Dunnet's test was used to determine the statistical significance of different groups. The difference was considered statistically significant when p < 0.05 .

3.1 Cell viability assay of BV-2 microglia and TNC-1 astrocytes

The cytotoxicity of Gastrodin on BV-2 microglia and Azilsartan on TNC-1 astrocytes was assessed by the CCK8 assay which showed that Gastrodin in the range of 0–1mg/ml and Azilsartan in the range of 0-160µM did not result in significant toxic effect (Fig. 1).

3.2 TNC-1 activation is mediated by LPS-stimulated BV-2 microglia conditioned medium

Western blot was used to detect the protein expression of TNF-α in the normal control group, LPS group, CM group, and CM + LPS group (Fig. 2). The results showed that TNC-1 astrocytes in the normal group hardly expressed TNF-α, and there was no statistically significant difference between the LPS group and the normal group. The CM group expressed a moderate amount of TNF-α, while the TNF-α protein expression in the CM + LPS group was significantly increased. The results indicate that TNC-1 activation was only mildly elicited and directly by LPS; instead, its robust activation is mediated by activated microglia.

3.3 Gastrodin reduced TNF-α expression and increased BDNF and IGF-1 expression in activated TNC-1 astrocytes via BV-2 microglia

Western blot was used to detect the protein expression changes of TNF-α, BDNF and IGF-1 in the CM group, CM + LPS group and low (0.17mM) and high (0.34mM) doses of Gastrodin treatment groups (Fig. 3). The results showed that TNF-α, BDNF and IGF-1 expression in TNC-1 astrocytes incubated with the BV-2 conditioned medium (CM) was negligible. The expression level of TNF-α and BDNF was significantly increased after treatment with CM + LPS when compared with the control in CM; likewise, TNC-1 incubated in CM + LPS exhibited moderate IGF-1 expression than that in CM. When treated with CM + LPS + G_L and CM + LPS + G_H, the expression of BDNF and IGF-1 was further elevated, whereas that of TNF-α was decreased as compared with that of CM + LPS treated cells. BDNF immunofluorescence in TNC-1 incubated in CM + LPS was increased in parallel to Western blot. The fluorescence intensity of IGF-1 and BDNF after Gastrodin treatment was further upregulated than that of the CM + LPS group. Expression changes of the respective proteins are indicated against the corresponding bar graphs. (Fig. 4).

3.4 Gastrodin decreased ATO and AT ₁ expression but promoted SirT3 expression in activated TNC-1 astrocytes via BV-2 microglia

Western blot and double immunofluorescence labeling were used to detect the expression of ATO, AT₁ and SirT3 proteins in the CM group, CM + LPS group, CM + LPS + G_L (0.17mM) group and CM + LPS + G_H (0.34mM) group. Western blot showed that the protein expression of ATO, AT₁ and SirT3 was enhanced in TNC-1 treated with CM + LPS. When treated with low and high doses of Gastrodin, the expression level of ATO, AT₁ was downregulated, whereas that of SirT3 was further enhanced as compared with that of CM + LPS treated cells (Fig. 3). ATO, AT₁ and SirT3 immunofluorescence in TNC-1 incubated in CM + LPS was increased in parallel to Western blot. Meanwhile, the expression of ATO and AT₁ decreased after Gastrodin treatment, while the expression of SirT3 was further enhanced (Fig. 5).

3.5 Gastrodin promoted S100A10 expression but inhibited C3 expression in activated TNC-1 astrocytes via BV-2 microglia

By Western blot, the protein expression changes of C3 and S100A10 in the CM group, CM + LPS group and low (0.17mM) and high (0.34mM) doses of Gastrodin treatment group were determined (Fig. 3). The results showed that C3 protein expression was significantly upregulated in CM + LPS group; however, its expression was significantly decreased when treated with two different doses of Gastrodin, when compared with the CM + LPS group. In addition, S100A10 expression was significantly decreased in the CM + LPS group compared with the CM group; on the contrary, following pretreatment with Gastrodin at concentrations 0.17 mM, the expression of S100A10 was enhanced, when compared with the CM + LPS group. This indicated that Gastrodin could inhibit A1 astrocytes and promote the expression of A2 astrocytes.

3.6 Inhibition of AT ₁ with Azilsartan suppressed the expression of TNF-α and promoted SirT3 expression; also, AT₁ receptor was involved in reactive astrocyte phenotype

Western blot showed that the expression level of TNF-α was decreased in the CM + LPS + A group, whereas that of SirT3 was significantly increased compared with the CM + LPS group. In comparison to the CM group, the expression of Sirt3 was also increased in CM + A group. Meanwhile, compared with the CM + LPS + G_L group, the expression of TNF-α was reduced after treatment with Azilsartan (CM + LPS + A + G_L). This suggests that AT₁ inhibitor (Azilsartan) can reduce the expression of inflammatory factors but promote the expression of SirT3 in reactive astrocytes induced by activated microglia. Very interestingly, compared with CM + LPS + A group, the expression of TNF-α was further suppressed in CM + LPS + A + G_L group. However, the combination of AT₁I and Gastrodin did not exert a cumulative effect on the expression of SirT3 (Fig. 6). The results indicated that Gastrodin could enhance the expression of SirT3 through AT₁ receptors. On the other, the inhibitory effect of Gastrodin on inflammation may not be exclusively through AT₁ receptors, and indeed other pathways may be involved.

In addition, C3 expression was decreased while the expression of S100A10 was increased in Azilsartan treated group (CM + LPS + A) when compared with the CM + LPS group. Remarkably, the combination of AT₁I and gastrodin could further promote the expression of S100A10 (Fig. 6). The results when taken together suggest that AT₁ receptors are involved in the phenotypic changes of reactive astrocytes, and that Gastrodin can affect or shift the phenotypes of reactive astrocytes through AT₁ receptors.

Astrocytes are one of the most abundant glial cell types in the CNS and play a key role in maintaining its health and function^[30]. When the CNS is injured, astrocytes undergo rapid changes in morphology and function, called reactive astrocytes^{[31, 32]}. It has been suggested that TNF-α secreted by microglia plays a key role as paracrine signals in regulating the neuroprotective function of astrogliosis^[33]. Meanwhile, microglia responses in traumatic brain injury (TBI) dynamically alter astrocyte responses to TBI. Among them, microglia-derived pro-inflammatory cytokines play a central role, which makes astrocytes reactive and acquire neuroprotective abilities^[19]. LPS is known to have a strong effect on microglial activation and is well suited to study the expression of various inflammatory mediators. Therefore, in this study, we cultured astrocytes with conditioned medium from LPS-stimulated microglia for subsequent experimental studies. Our results showed that TNF-α expression in untreated TNC-1 astrocytes was very low, and when TNC-1 astrocytes were directly exposed to LPS stimulation, up-regulation of TNF-α expression was not evident. However, TNF-α expression was significantly increased after culturing astrocytes with conditioned medium from LPS-stimulated microglia. This confirms that microglia can induce the activation of astrocytes^[18].

It is well documented that Gastrodin plays a certain role in brain protection, which can effectively reduce the activation of astrocytes after subarachnoid hemorrhage^[34], and inhibit the excessive autophagy and apoptosis of astrocytes induced by LPS^[35]. In addition, reactive astrocytes express certain levels of trophic factors, such as IGF-1^[36] when brain injury occurs. Some studies have found that injection of IGF-1 can play a certain neuroprotective effect in cerebral ischemia and ischemia-reperfusion models^{[37, 38]}. The present results have shown that the expression of BDNF, IGF-1 and TNF-α in TNC-1 astrocytes activated by microglia conditioned medium was significantly up-regulated when compared with the control group. However, in Gastrodin pretreatment cell gruop, the expression of BDNF and IGF-1 in TNC-1 astrocytes was further increased, but the expression of TNF-α was decreased. This indicates that Gastrodin can promote the activation of astrocytes and release of neurotrophic factors by them was mediated by microglia. However, the expression of inflammatory factors such as TNF-α was decreased, thereby confirming the anti-inflammatory propensity of Gastrodin.

The renin-angiotensin system (RAS) is an important humoral regulation system in the human body. It plays an important role in maintaining the relative stability of the internal environment in the human body. The different components of the RAS are expressed in many organs, notably the heart, kidneys, and brain^[39].The local/paracrine renin-angiotensin system (RAS) plays a major role in inflammatory processes in the peripheral tissues and the brain^[40]. In view of this, we had considered whether activated microglia-mediated release of inflammatory factors from astrocytes would be related to the renin-angiotensin system. AngII is an important effector peptide in the angiotensin system^[41] and exerts vascular remodeling effects by promoting cell proliferation, fibrosis, oxidative stress and inflammation^{[42, 43]}. Meanwhile, angiotensin II (Ang II) is the main effector of RAS, which has at least two receptor subtypes, called angiotensin II type 1 (AT₁) and angiotensin II type 2 (AT₂) receptors, but most of the classical effects are mediated by the Ang II type 1 receptor (AT₁R)^{[44, 45]}. Astrocytes are known to be the primary source of the precursor proteins angiotensinogen (ATO) and angiotensin II in the brain^[40]. Studies have found that most of the synthesis of ATO in the brain is produced by astrocytes^{[46, 47]}. At the same time, type 1 and type 2 Ang II receptors and two forms of angiotensin-converting enzyme were also found in astrocytes. Taken together, astrocytes possess a complete renin-angiotensin system^[48–50].Our results have shown that ATO and AT₁ expressions was significantly increased in astrocytes activated by microglia conditioned medium compared with the control group. However, the expression of both proteins was decreased after Gastrodin treatment. This suggests that Gastrodin can exert its anti-inflammatory effect by inhibiting the expression of ATO and AT₁ in reactive astrocytes induced by activated microglia.

Additionally, studies have found that astrocytes expressed AT₁ in the dentate gyrus after the lesion, and the number of infiltrating macrophages increased significantly after blocking AT₁^[51]. Ang II directly triggers apoptosis of dopaminergic neurons by activating AT₁ receptors in the rat substantia nigra. Azilsartan, an inhibitor of AT₁R, has been reported to ameliorate apoptosis of dopaminergic neurons and rescue characteristic Parkinson's disease behaviors in a rat model of Parkinson's disease (PD)^[52]. Furthermore, mitochondria-dependent apoptosis signaling is also involved in Ang II-induced apoptosis of dopaminergic neurons^[53]. SirT3, as a major protein deacetylase in mitochondria, is involved in the regulation of cellular redox, mitochondrial energy, biogenesis, kinetics, and apoptosis^[54–57]. Our previous study found that Gastrodin can increase Sirt3 expression in activated microglia^{[8, 9]}. We showed here that SirT3 expression in activated astrocytes induced by microglia conditioned medium followed the same trend. This suggests that Gastrodin can also promote the expression of SirT3 in astrocytes induced by activated microglia. In addition, it was reported that Ang II reduces SirT3 expression via AT₁, whereas AT₁ receptor antagonists upregulate SirT3.Overexpression of AT₁ and AT₂ in rat substantia nigra, dopaminergic neurons, and microglia was induced using a SirT3-specific inhibitor^[58]. Additionally, studies have found that Azilsartan can attenuate oxidative damage in brain endothelial cells by modulating mitochondrial function and inflammatory responses in vitro^[59]. Reducing Ang II and inhibiting AT₁R can reduce the production of pro-inflammatory factors TNF-α and IL-1β^[60]. The above studies have given some clues to the interrelation between the AT₁ receptor and SirT3. To explore the relationship between AT₁ and SirT3 in activated astrocytes as well as the effect of Gastrodin intervention, the expression of SirT3 along with TNF-α was detected after blocking AT₁R with Azilsartan. We have shown that SirT3 expression was significantly increased in astrocytes activated by microglia conditioned medium, and that it was further increased after blocking AT₁R. Meanwhile, TNF-α expression was significantly down-regulated after Azilsartan treatment. It is therefore suggested that AT₁-SirT3 signaling pathway plays an important role in the inflammatory response of activated astrocytes. Ang II can inhibit the expression of SirT3 through AT₁ receptor in activated astrocytes, and affect cell survival. Very strikingly, the combination of Azilsartan and Gastrodin can further down-regulate the expression of TNF-α, yet the expression of SirT3 remained unchanged. These results indicate that Gastrodin can up-regulate SirT3 protein by inhibiting AT₁ and exert its neuroprotective effect.

Astrocytes can be divided into A1 type and A2 type according to different stimulatory factors^{[18, 61]}. A1/A2 astrocytes with varying degrees of polarization are expressed in normal aging^[62], acute brain injury^[63], and neurodegenerative diseases such as Parkinson's disease (PD)^[64] and Alzheimer's disease (AD)^[61]. Transcriptome analysis of reactive astrocytes found that genes such as C3 and H2-D1 were preferentially expressed in LPS-induced A1-type reactive astrocytes, while S100 calcium binding protein A10 (S100A10), pentraxin-3 (PTX3) among others are preferentially expressed in MCAO-induced A2-type reactive astrocytes^[65]. A1 astrocytes may secrete certain neurotoxins that kill a subset of neurons and mature oligodendrocytes. Conversely, A2 astrocytes appear to upregulate the expression of neurotrophic or anti-inflammatory factors that promote neuronal survival and growth and support repair function^[66]. At the same time, it has been reported that A1-reactive astrocytes can be induced by cytokines secreted of activated M1-type microglia, which has a neurotoxic function^[18]. Our results showed that the expression of C3 (A1 astrocytes marker) in the CM + LPS group was significantly higher than that of the control group, while the expression of S100A10 (A2 astrocytes marker) was significantly lower than that of the control group. This further supports that activated microglia can affect the phenotypic changes in reactive astrocytes. Another point to note was that the C3 expression was significantly decreased with Gastrodin treatment, whereas S100A10 expression was significantly increased. This lends further support to the notion that Gastrodin could promote the expression of A2-type reactive astrocytes and inhibit the expression of A1-type reactive astrocytes. Interestingly, in TNC-1 astrocytes treated with the conditioned medium from microglia stimulated with LPS, the expression of C3 and S100A10 was significantly reversed after Azilsartan treatment. Arising from the above results, it can be confidently concludecd that the phenotypes of reactive astrocytes are regulated by the AT₁R. Meanwhile, the combination of Gastrodin and Azilsartan can further amplify the expression of S100A10 compared with CM + LPS + A group. Thus, Gastrodin can promote the polarization of astrocytes towards A2 phenotype through AT₁R.

The present results have shown that the expression of angiotensin system-related proteins (ATO and AT₁), SirT3, TNF-α, IGF-1 and BDNF, and the marker C3 of A1 astrocytes was significantly increased in reactive astrocytes by LPS-stimulated BV-2 conditioned medium. In contrast, the S100A10 expression, a marker for A2-type astrocytes, was decreased. In addition, blocking of AT₁ receptors with Azilsartan can effectively inhibit the expression of C3 and TNF-α in reactive astrocytes, but promote the expression of S100A10 and SirT3. We have further shown that Gastrodin can regulate the expression of the various biomarkers in reactive astrocytes through the AT₁-SirT3 signaling pathway. More importantly, it promotes the expression of SirT3 and IGF-1 and BDNF, thus exerting neuroprotective effects. The activation of astrocytes and the protective effect of Gastrodin may be mediated by microglia, thus underscoring a crosstalk relationship between the glial cell types (Fig. 7). However, the molecular mechanism of Gastrodin-mediated activation of astrocytes by microglia remains to be defined.

HIBD

hypoxic-ischemia brain damage

SirT3

Sirtuin3

AT₁

angiotensin II type 1

AT₂

angiotensin II type 2

RAS

Renin angiotensin system

LPS

lipopolysaccharide

conditioned medium

ATO

angiotensinogen

Ang II

Angiotensin II

AT₁R

angiotensin II receptor subtype AT₁

CNS

central nervous system

nitric oxide

COX2

cyclooxygenase 2

TNF-α

the tumor necrosis factor-α

IL-1β

interleukin-1β

IL-1α

interleukin-1α

BDNF

brain derived neurotrophic factor

IGF1

insulin-like growth factors − 1

complement protein C3

S100A10

S100 calcium binding protein A10

CCK8

cell counting Kit-8

DMEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

BCA

Bicinchoninic acid

ROS

reactive oxygen species

Competing interests

The authors declare that they have no competing interests.

Author Contributions

J-JL conceptualized and designed this study. J-JL and MZ supervised the whole project. H-JZ, X-QR, KG and P-XW carried out the experiments. H-JZ and X-QR, performed acquisition and analysis of data. H-JZ and J-JL prepared the manuscript. All authors read and approved the final manuscript.

Funding

This project was supported by grants from National Natural Science Foundation of China (No. 31960194, 31460274, J-J Li) and Applied Basic Research Projects of Yunnan Province of China (No. 2019FE001 (-003), J-J Li).

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No competing interests reported.

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Gastrodin regulates expression of angiotensin-SirT3 and proinflammatory mediators in reactive astrocytes via activated microglia

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 BV-2 microglia and TNC-1 astrocyte cultures

2.2 Cell viability assay of BV-2 microglia and TNC-1 astrocytes

2.3 Preparation and use of BV‑2 conditioned medium

2.4 Treatment of TNC-1 astrocytes with Azilsartan

2.5 TNC1 astrocytes and treatment with BV‑2 conditioned medium

2.6 Western blot analysis of TNC-1 astrocytes

2.7 Immunofluorescence labeling of TNC-1 astrocytes

2.8 Statistical analysis

3. Results

3.1 Cell viability assay of BV-2 microglia and TNC-1 astrocytes

3.2 TNC-1 activation is mediated by LPS-stimulated BV-2 microglia conditioned medium

3.5 Gastrodin promoted S100A10 expression but inhibited C3 expression in activated TNC-1 astrocytes via BV-2 microglia

4. Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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