Hydrogen Sulfide Attenuates Lipopolysaccharide-Induced Inflammation via the P-glycoprotein and NF-κB Pathway in Astrocytes

Abstract

Astrocyte activation is key in neurodegenerative diseases. Hydrogen sulfide (H₂S) exhibits neuroprotective effects on astrocytes, although the underlying molecular mechanism remains unclear. Here, we explored the effects of H₂S on lipopolysaccharide (LPS)-induced astrocyte activation and astrocyte-mediated neuroinflammation. After inducing primary astrocytes via LPS exposure, H₂S levels were altered. The generation and secretion of inflammatory mediators by astrocytes and their interrelation with P-glycoprotein (P-gp), an important transporter belonging to the ABC transporter family, were assessed. Activated astrocytes showed upregulated glial fibrillary acidic protein (GFAP) mRNA expression, and significantly increased proinflammatory factor mRNA/protein expression and release. The secretory capacity of astrocytes was reduced, with significantly decreased proinflammatory factor levels in culture supernatant after P-gp inhibitor verapamil pretreatment. The increase in the intracellular H₂S level inhibited LPS-induced GFAP expression and P65 nuclear entry in astrocytes. mRNA expression and release of proinflammatory factors were reduced significantly, with no significant changes in cytoplasmic protein expression. S-sulfhydration levels increased significantly with the increased concentration of sodium hydrosulfide or S-adenosyl-L-methionine addition, with only moderate changes in astrocyte P-gp expression. H₂S regulates NF-κB activation, leads to S-sulfhydration of P-gp, and inhibits the biosynthesis and secretion of proinflammatory factors by astrocytes. The regulatory effects of H₂S on astrocytes may have clinical value for exploring new therapeutic strategies against neurodegenerative diseases.

Keywords: Astrocyte; Hydrogen sulfide; NF-κB; Neuroinflammation; P-glycoprotein.

Fig. 1

P-gp regulates the extracellular secretion of proinflammatory factors in primary astrocytes stimulated by LPS. LPS-stimulated proinflammatory cytokine (IL-1β, IL-6, and TNF-α) expression and extracellular secretion in primary astrocytes were suppressed following administration of the P-gp inhibitor verapamil (70 μM). a RT-PCR analysis of mRNA expression of proinflammatory factors in astrocytes. b ELISA to detect proinflammatory factor levels in the cell culture supernatant. c Western blotting to detect proinflammatory factor levels in the cytoplasm. d Rhodamine 123 (Rho123) accumulation assay to detect P-gp function: green fluorescence for intracellular Rhodamine 123, blue fluorescence for nucleus. A, Con; B, LPS; C, verapamil; D, LPS + verapamil. n = 3. **p < 0.01 vs Con, ***p < 0.001 vs Con; ##p < 0.01 vs LPS, ###p < 0.001 vs LPS

Fig. 2

Exogenous H₂S supplementation inhibited the LPS-induced production and secretion of inflammatory factors by primary astrocytes. LPS-stimulated GFAP mRNA expression, proinflammatory cytokine (IL-1β, IL-6, and TNF-α) expression and extracellular secretion in astrocytes were suppressed following administration of NaHS (50, 100, and 300 µM). a RT-PCR of GFAP mRNA expression in primary astrocytes. b RT-PCR analysis of proinflammatory factor mRNA expression in primary astrocytes. c ELISA of proinflammatory factor levels in the cell culture supernatant. d Western blotting analysis of cytoplasmic proinflammatory factor levels. A, Con; B, LPS; F, LPS + NaHS 50 µM; G, LPS + NaHS 100 µM; H, LPS + NaHS 300 µM. n = 3. **p < 0.01 vs Con, ***p < 0.001 vs Con; ##p < 0.01 vs LPS, ###p < 0.001 vs LPS

Fig. 3

NaHS inhibited the LPS-induced activation of the NF-kB pathway in astrocytes. The nuclear entry of LPS-stimulated P65 was inhibited by NaHS, and the inhibition increased with increasing NaHS concentration. The distribution of P65 in the nucleus and cytoplasm of astrocytes was detected via western blotting. A, Con; B, LPS; F, LPS + NaHS 50 µM; G, LPS + NaHS 100 µM; H, LPS + NaHS 300 µM. n = 3. *p < 0.05 vs Con, **p < 0.01 vs Con, ***p < 0.001 vs Con; #p < 0.05 vs LPS, ##p < 0.01 vs LPS

Fig. 4

NaHS regulates astrocyte secretory activity through inhibiting P-gp-mediated transport. The enhanced transport of P-gp in LPS-stimulated astrocytes was inhibited by NaHS, as shown by the accumulation of Rhodamine 123 (Rho123) in astrocytes, which was inversely proportional to the concentration of NaHS. Green fluorescence for intracellular Rhodamine 123, and blue fluorescence for the nucleus. A, Con; B, LPS; C, NaHS 50 µM; D, NaHS 100 µM; E, NaHS 300 µM; F, LPS + NaHS 50 µM; G, LPS + NaHS 100 µM; H, LPS + NaHS 300 µM. n = 3. *p < 0.05 vs Con, **p < 0.01 vs Con, ***p < 0.001 vs Con; #p < 0.05 vs LPS, ###p < 0.001 vs LPS

Fig. 5

Exogenous H₂S supplementation increases astrocyte P-gp sulfation without affecting its expression at the mRNA and protein level. a RT-PCR analysis of astrocytic P-gp-encoding gene expression. b P-gp was S-sulfhydrated by NaHS (300 µM), detected via modified biotin switch (S-sulfhydration) assay. c Biotin switch (S-sulfhydration) analysis to detect the level of P-gp sulfation in astrocytes. A, Con; B, LPS; F, LPS + NaHS 50 µM; G, LPS + NaHS 100 µM; H, LPS + NaHS 300 µM. n = 3. DTT, DL-Dithiothreitol; SHY-Pgp, sulfhydrated Pgp; SHY-Tubulin, sulfhydrated Tubulin. *p < 0.05 vs Con, **p < 0.01 vs Con; #p < 0.05 vs LPS

Fig. 6

Effects of endogenous H₂S on the inflammatory activation of astrocytes and the LPS-induced production and secretion of inflammatory factors by astrocytes. Upon LPS stimulation, GFAP mRNA expression and proinflammatory cytokine (IL-1β, IL-6, and TNF-α) expression and secretion in primary astrocytes were suppressed following administration of SAM, which could be eliminated by the addition of the CBS inhibitor AOAA. a RT-PCR of GFAP mRNA expression in primary astrocytes. b RT-PCR of proinflammatory factor expression in primary astrocytes. c ELISA of proinflammatory factor levels in the cell culture supernatant. d Western blotting analysis of cytoplasmic proinflammatory factor levels. A, Con; B, SAM; C, AOAA; D, LPS; E, LPS + SAM; F, LPS + SAM + AOAA; G, LPS + AOAA. n = 3. **p < 0.01 vs Con, ***p < 0.001 vs Con; #p < 0.05 vs LPS, ##p < 0.05 vs LPS, ###p < 0.001 vs LPS

Fig. 7

Effects of endogenous H₂S on the NF-kB pathway of LPS-induced astrocytes. The nuclear entry of LPS-stimulated P65 was inhibited by SAM, which could be inhibited by the addition of AOAA. The distribution of P65 in the nucleus and cytoplasm of astrocytes was detected by western blotting. A, Con; B, SAM; C, AOAA; D, LPS; E, LPS + SAM; F, LPS + SAM + AOAA; G, LPS + AOAA. n = 3. *p < 0.01 vs Con, **p < 0.01 vs Con, ***p < 0.001 vs Con; ##p < 0.05 vs LPS, ###p < 0.001 vs LPS

Fig. 8

The effect of endogenous H₂S on the function of P-gp in astrocytes. SAM inhibited basal and LPS-activated P-gp transport activity in astrocytes, while AOAA reversed or enhanced P-gp transport activity in astrocytes. Green fluorescence for intracellular Rhodamine 123, and blue fluorescence for the nucleus. Rhodamine 123 (Rho123) accumulation assay was used to detect the transport function of P-gp in astrocytes. A, Con; B, SAM; C, AOAA; D, LPS; E, LPS + SAM; F, LPS + SAM + AOAA; G, LPS + AOAA. n = 3. **p < 0.01 vs Con, ***p < 0.001 vs Con; ###p < 0.001 vs LPS

Fig. 9

Endogenous H₂S affects P-gp sulfation in astrocytes without affecting its expression at mRNA and protein levels. a RT-PCR of P-gp-encoding gene expression in astrocytes. b Biotin switch (S-sulfhydration) analysis to detect the level of P-gp sulfation in astrocytes. A, Con; B, SAM; C, AOAA; D, LPS; E, LPS + SAM; F, LPS + SAM + AOAA; G, LPS + AOAA. n = 3. **p < 0.01 vs Con, ***p < 0.001 vs Con; ##p < 0.001 vs LPS

Fig. 10

Schematic representation of the results