Hydrogen sulphide ameliorates dopamine‐induced astrocytic inflammation and neurodegeneration in minimal hepatic encephalopathy

Abstract It has been demonstrated that the action of dopamine (DA) could enhance the production of tumour necrosis factor‐α (TNF‐α) by astrocytes and potentiate neuronal apoptosis in minimal hepatic encephalopathy (MHE). Recently, sodium hydrosulfide (NaHS) has been found to have neuroprotective properties. Our study addressed whether NaHS could rescue DA‐challenged inflammation and apoptosis in neurons to ameliorate memory impairment in MHE rats and in the neuron and astrocyte coculture system. We found that NaHS suppressed DA‐induced p65 acetylation, resulting in reduced TNF‐α production in astrocytes both in vitro and in vivo. Furthermore, decreased apoptosis was observed in neurons exposed to conditioned medium from DA + NaHS‐challenged astrocytes, which was similar to the results obtained in the neurons exposed to TNF‐α + NaHS, suggesting a therapeutic effect of NaHS on the suppression of neuronal apoptosis via the reduction of TNF‐α level. DA triggered the inactivation of p70 S6 ribosomal kinase (S6K1) and dephosphorylation of Bad, resulting in the disaggregation of Bclxl and Bak and the release of cytochrome c (Cyt. c), and this process could be reversed by NaHS administration. Our work demonstrated that NaHS attenuated DA‐induced astrocytic TNF‐α release and ameliorated inflammation‐induced neuronal apoptosis in MHE. Further research into this approach may uncover future potential therapeutic strategies for MHE.

pathological processes of neurodegenerative disorders such as vascular dementia, Alzheimer's disease (AD) and Parkinson's disease (PD). 5 Meanwhile, it was reported that the intervention of H 2 S could ameliorate the learning and memory impairment and neuroinflammation 7 by specifically enhancing hippocampal long-term potentiation. 8 NaHS, as an exogenous donor of H 2 S, prevents inflammatory response in post-ischaemic murine small intestine 9 and inhibits apoptosis by reducing caspase-3 expression in the mouse brain. 10 Brain dopamine (DA) has been implicated in cognitive processes such as working memory and cognitive flexibility, 11 and the DA system is involved in cognitive decline. 12 Our previous studies have confirmed that the pathogenesis of minimal hepatic encephalopathy (MHE) was caused by the abnormal elevation of intracranial DA. [13][14][15][16][17][18] It has been demonstrated as a novel regulatory mechanism that DAinduced astrocytic tumour necrosis factor-α (TNF-α) release triggers progressive neurodegeneration, which leads to learning and memory decline in MHE. 19 Therefore, we suggested that NaHS might have therapeutic effects on DA-induced cognitive decline by reducing the secretion of astrocytic TNF-α in MHE rats.
In this study, we mainly focused on investigating whether NaHS had dual effects on the DA-induced MHE. Firstly, we examined whether NaHS attenuated DA-mediated TNF-α release from astrocytes via p65 signalling. Secondly, we assessed whether NaHS reversed TNF-α induced neuronal apoptosis. week for 8 weeks to induce cirrhosis. Rats treated with TAA exhibited reduced exercise capacity, and rats with lethargy and delayed development of coma were classified as HE. 16 The MHE status was determined by WFT values above average ±1.96 SD or YM values below average ±1.96 SD after TAA treatment in asymptomatic rats.

| DA-treated rat models and treatments
Intraventricular injection of DA hydrochloride (10 μg/3 μL in saline) into the left ventricle of anaesthetized rats (anterior and posterior 0.3 mm; lateral 1.0 mm; horizontal, anterior reg 3.0 mm), one time a week for 3 weeks (n = 15). Intraperitoneal (i.p.) injection of NaHS (6 mg/kg) was performed one time a week after the last DA injection for 3 weeks. When the rats were subjected to the YM and WFT tests after the last injection, samples of the killed rats were collected.

| Behavioural tests
Arbitrarily placing the rat in the end of the arm in the three-armed device allowed the rat to freely discover the maze for 8 minutes.
The percentage of spontaneous alternation (SA%) was determined by the ratio of arm selection to total selection. 20 The rat placed in the upper right corner of the WFT instrument was arranged to find and drink the water in the wall within 3 minutes. The elapsed time of entering into the alcove (entry latency, EL), elapsed time of first touching/smelling/licking of the water pipe (contacting latency, CL) and elapsed time of starting drinking from the water pipe (drinking latency, DL) were carried out. 21

| Determination of DA levels
Serum or lysates of the liver/hippocampus were then collected and analysed for quantitative determination of DA using a commercial DA ELISA assay kit (Cusabio, Wuhan, China). Data analysis was performed by microtiter plate reader (BIO-TEK Synergy 2). The results presented are from three independent experiments (n = 3).

| Culture and treatments of primary hippocampal and cortical astrocytes (PHAs and PCAs)
Primary hippocampal astrocytes (PHAs) or Primary cortical astrocytes (PCAs) were isolated from hippocampus or cerebral cortex tissues of 1-day-old SD rat pups by mechanical digestion. 11 mL of 1% serum-containing DMEM/ F12 medium was used to incubate cells seeded in 75 cm 2 tissue culture flasks at a density of 15 × 10 6 cells for 72 hours. The medium was changed every 72 hours. After 7 days of incubation in the primary culture, oligodendrocytes were separated from astrocytes by shaking at 200× g for 18 hours at 37°C. Cells were plated in a six-well plate coated with poly-L-lysine after incubation for 7 days in a new 75 cm 2 flask. Treatment of astrocytes with NaHS (50, 100 or 300 μmol/L) was performed in a pre-incubation of DA (final concentration 10 μmol/L) or TNF-α (final concentration of 1 ng/mL) for 24h. After changing the medium with serum-free DMEM/ F12 for 6-8 hours, conditioned glial medium (GCM) was collected. The collected GCM was centrifuged at 1000× g for 4 minutes, and then 10 times concentrated with the Amicon Ultra-4 filter device (Millibo, Billy Rica, MA, USA).

| Culture and treatments Primary hippocampal and cortical neurons (PHNs and PCNs)
PHNs or PCNs were gained from the hippocampus or cerebral cortex of 1-day-old SD rat pups. The tissue was mechanically digested in medium containing trypin and DNase, and cells at the density of 2 ×

| ELISA assay
Tumour necrosis factor-α levels in 96-well plates were detected using a high-sensitivity sandwich ELISA kit using a Thermo Fisher Multiskan MC plate reader for spectrophotometry in accordance with the instructions from Promega and SydLabs.  Table 1.

| Immunoblotting
Homogenized tissues or cells were lysed by using RIPA lysis buffer

| Double immunofluorescence staining and double Staining of TUNEL and NeuN
Brain slices or cell coverslips were fixed with 4% paraformaldehyde for 30 minutes, and the cell membrane was permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. 5% normal goat serum in PBS was used for blockade for 1 hour at room tempera-

| Statistical analysis
The expression of data was shown as mean ± SD. Analysis of data

| NaHS improved DA-induced cognitive impairment in MHE rats
Our goal here is to investigate the potential and beneficial effects of NaHS on DA-triggered astrocytic TNF-α release and neuronal toxicity in vitro and in vivo. First, we assessed H 2 S content in MHE rats and DA-treated rats after NaHS administration. We observed that low dose NaHS (1 or 3 mg/kg) treatment resulted in increased trend of H 2 S production compared to MHE or DA-treated group; however, H 2 S production was significantly increased in both of MHE-and DAtreated groups after high dose of NaHS treatments ( Figure 1A).
Then, we tested whether NaHS improved memory loss in MHE rats and DA-treated rats. In YM, administration of NaHS increased SA% in MHE rats and DA-treated rats in a dose-dependent manner ( Figure 1B). In WFT, NaHS treatment (6 mg/kg) induced significant decreases in EL, CL and DL in MHE rats and DA-treated rats ( Figure 1C). These data indicate that NaHS rescues DA-triggered memory decline in MHE. We also confirmed that DA levels in the brains were obviously increased in MHE rats, whereas NaHS administration significantly reduced DA levels ( Figure 1D). Of note, our study presents that H 2 S is sufficient to ameliorate DA-driven memory impairment by inhibiting inflammation and neuronal apoptosis.

| NaHS decreased DA-mediated astrocytic TNF-α release and p65 acetylation in vitro
We first examined our cell culture by RT-PCR using targeting specific cell markers including astrocytes (GFAP), endothelial cells (CD31), microglia (IBA1), neurons (MAP2) and oligodendrocytes (O 4 ). We found that PHAs (Figure 2A) or PCAs ( Figure 2B) had higher expressions of GFAP and lower expressions of other cell type specific genes compared with whole brain. Altogether, these data displayed successfully enriched astrocyte cultures. Because the potential toxicity of NaHS for astrocytes has never been evaluated, we then examined whether NaHS affected cell death by the MTT assay. 0-300 μmol/L NaHS did not exhibit any toxic effects on the PHAs viability ( Figure 2C).
Dopamine has previously been reported to induce TNF-α release from astrocytes. 19 Thus, we tested the impact of NaHS on DA-induced TNF-α release from astrocytes in vitro. By IB analysis, NaHS treatment markedly reduced TNF-α release in a dose-dependent manner from The transcription factor nuclear factor kappa-B (NF-kB) played a key role in regulating inflammatory activity by the acetylation of NF-kB subunit p65/RelA. 22 We investigated whether NaHS decreased TNF-α production by inhibiting p65 acetylation. IB analysis showed that DA significantly increased p65 acetylation in PHAs, while high dose of NaHS treatment obviously reversed the effect of DA, compared with unstimulated cells ( Figure 2M,N). The increased p65 acetylation induced by DA treatment in PCAs was blocked by the addition of high dose of NaHS ( Figure 2O,P). Our study showed that NaHS treatment could markedly inhibit DA-induced pro-inflammatory cytokine release.

| NaHS suppressed TNF-α-induced neuronal apoptosis in vitro
It has been reported that neurodegeneration in MHE is triggered by DA depending on astrocytic TNF-α mediation. 19 We first tested our cell culture through RT-PCR using targeting specific cell markers.
We found that PHNs ( Figure 3A) or PCNs ( Figure 3B) had higher expressions of MAP2 and lower expressions of other cell type specific genes compared to the whole brain. Altogether, these data showed successfully enriched neuron cultures. We then examined the potential toxicity of NaHS on cell viability of neurons by the MTT assay, but 0-300 μmol/L NaHS had no effect on the PHNs death ( Figure 3C).
Tumour necrosis factor-α, not DA, was reported to directly induce neuronal apoptosis. 19 Thus, we tested whether NaHS had an effect on TNF-α-stimulated neuronal apoptosis by TUNEL staining and immunoblotting using caspase 3/9 and Cyt. c. Based on MTT assay, PHNs Cyt. c release to cytoplasm in PHNs, whereas GCM PHAs-DA-300 NaHS treatment reduced Cyt. c release ( Figure 3J,K). We also observed increased cytoplasmic Cyt. c in PCNs with GCM PCAs-DA treatment, which was dramatically reversed by the GCM PCAs-DA-300 NaHS treatment ( Figure 3L,M). These data confirmed that NaHS ameliorated apoptosis by reduction of TNF-α level. We next investigated whether NaHS directly attenuated TNF-αinduced neuronal apoptosis. MTT assay showed that TNF-α significantly induced cell death in PHNs and NaHS abrogated the effect of TNF-α ( Figure 4A). PCNs showed a significant elevated signal by TUNEL staining in response to TNF-α, which was abolished by NaHS ( Figure 4B). As determined by the IB analysis in Figure 4C,D, we confirmed an up-regulated Caspase3/9 expression in PHNs exposed to TNF-α and NaHS addition decreased the Caspase3/9 expression in a dose-dependent manner. Caspase3 expression was also elevated in PCNs exposed to TNF-α, which was blocked by the addition of 300 μmol/L NaHS ( Figure 4E,F). Based on qPCR, TNF-α treatment increased Caspase3/8/9 mRNA in PHAs or PCAs, and NaHS treatment reversed the effect of TNF-α in a dose-dependent fashion ( Figure 4G). Tumour necrosis factor-α stimulated mitochondria to secrete cellular Cyt.c to the cytoplasm, which was dramatically reversed by the 300 μmol/L NaHS treatment in PHNs ( Figure 4H,I) or PCNs ( Figure 4J,K). We confirmed that the treatment with GCM PHAs-DA-300NaHS or GCM PCAs-DA-300NaHS , in which TNF-α content was significantly decreased by NaHS, inhibited apoptosis of neurons.

| NaHS blocked TNF-α-induced dephosphorylation of Bad via S6K1 in neurons
S6K1 might have been implicated in regulating cell death and survival. 23 Here, we investigated whether NaHS affected S6K1's phosphorylation. However, as determined by IB analysis, we found decreased phosphorylation of S6K1 in PHNs with TNF-α treatment, while 300 μmol/L NaHS induced the reversal of the effect of TNF-α ( Figure 5A,B). We further found that the addition of NaHS also in- Bad, as a pro-apoptotic member of BCL-2 family, once Phosphorylated, was involved in the maintenance of cell survival. 24,25 We assessed whether NaHS elicited phosphorylation of Bad through phosphorylation cascade of S6K1 using S6K1 inhibition and siR-NA-induced silencing. We first tested the efficiency of S6K1 siRNA transfection into MHE astrocytes. As determined by qPCR, PHNs showed weak S6K1 after S6K1 siRNA transfection ( Figure 5G), as  Figure 5N,O).
NaHS induced the phosphorylation of Bad, and the interaction between Bclxl and pro-apoptotic protein Bak, resulting in the inhibition of mitochondria-mediated apoptosis.

| NaHS altered TNF-α-induced Bad-Bclxl-Bak interactions in neurons
The phosphorylation of Bad elicited the disruption of the binding of Bad to Bclxl and enhancement of the binding of Bak to Bclxl, leading to cell survival. [24][25][26][27] Thus, we assessed whether GCM PHAs-DA-300NaHS played a role in the association of Bak and Bclxl or Bclxl and Bad by co-immunoprecipitation. As determined in Figure 6A, GCM PHAs-DA treatment induced an increase in Bclxl level that co-immunoprecipitated with Bad, which was blocked by GCM PHAs-DA-300NaHS treatment.
GCM PHAs-DA treatment reduced the amount of Bak and increased the amount of Bad that co-immunoprecipitated with Bclxl, which was also abrogated by GCM PHAs-DA-300NaHS treatment. GCM PHAs-DA treatment also reduced the level of Bclxl that co-immunoprecipitated with Bak, which was also diminished by GCM PHAs-DA-300NaHS treatment. The results suggested that NaHS stimulated the Bak-Bclxl interaction and disrupted Bclxl-Bad interaction in neurons through reduction of TNF-α release from astrocytes.
Then, we addressed the impact of NaHS on TNF-α-mediated Bak-Bclxl or Bclxl-Bad interaction. As shown in Figure 6B, TNF-α increased Bclxl level that co-immunoprecipitated with Bad, which was blocked by NaHS. TNF-α also triggered decreased levels of Bax and increased level of Bad that co-immunoprecipitated with Bclxl, which was also diminished by NaHS. The level of Bclxl that co-immunoprecipitated with Bak was significantly decreased by TNF-α, which was also abated by NaHS.
Taken together, we suggested that NaHS protected against apoptosis, possibly through the phosphorylation of Bad, which induced dissociation of Bad from mitochondrial Bclxl followed by the interaction between Bak and mitochondrial Bclxl, and led to anti-apoptosis.

| NaHS attenuated DA-stimulated inflammatory response and neurodegeneration in MHE rats
We then assessed the impact of NaHS on the inflammatory signalling pathway in vivo. A significant increase in TNF-α expression was observed in the hippocampus of MHE and DA-treated rats, while NaHS treatment improved the expression of TNF-α ( Figure 7A,B). NaHS administration markedly reversed the levels of Caspase3/9, indicating an involvement of Caspase3/9 in reducing TNF-α level to reduce apoptosis. It is documented that NaHS suppressed DA-induced TNF-α production to inhibit neuronal apoptosis in astrocytes and in vivo.

| D ISCUSS I ON
Astrocytes are now emerging as pivotal regulators of potent proinflammatory responses in the central nervous system. 28,29 Several studies reported that DA overload-induced astrocytic TNF-α production promoted neuronal apoptosis, leading to the impairment of learning and working memory in the process of MHE pathologies. 18,19,21,30 Our goal here is to investigate the potential and beneficial effects of NaHS on DA-triggered astrocytic TNF-α release and neuronal toxicity in vitro and in vivo. We now found that the treatment with GCM PHAs-DA-300NaHS or GCM PCAs-DA-300NaHS , in which TNF-α content was significantly decreased by NaHS, suppresses the apoptosis of neurons. Of note, H 2 S is sufficient to reduce inflammation and neuronal apoptosis to ameliorate DA-driven memory impairment. We also presented here that NaHS directly inhibited An NF-κB subunit p65 can either potentiate or diminish NF-kB signalling by regulating the particular acetylated lysine residues, 22,[31][32][33] which is involved in producing pro-inflammatory cytokines to modulate the developments of age-related diseases. [33][34][35][36] Our data showed that DA promoted acetylation of RelA/p65 and increased pro-inflammatory cytokine levels in astrocytes. Once p65 is acetylated and activated, NF-ƙB will persistently be activated for the production of TNF-α in response to DA exposure, which could be reversed by NaHS treatment.  In summary, it is documented that NaHS suppressed DA-induced TNF-α production and p65 deacetylation to inhibit neuronal apoptosis in astrocytes and in vivo. Moreover, we also presented here that NaHS directly reduced TNF-α-mediated phosphorylation of S6K1/ Bad and inactivated the pro-apoptotic function of Bad induced by TNF-α in neurons and in vivo. Furthermore, our work provides novel insights into the anti-inflammatory and anti-apoptotic dual property of H 2 S for the treatment of MHE.

ACK N OWLED G EM ENT
This study was funded by Basic scientific research projects of Wenzhou (Y20180076) and the Natural Science Foundation of China (81671042, 81300308, 81171088 and 81371396).

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest. Project administration (equal); Supervision (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.