Pathophysiological Concepts in Multiple Sclerosis and the Therapeutic Effects of Hydrogen Sulfide

Abstract

Introduction: Multiple sclerosis (MS) is generally known as a manageable but not yet curable autoimmune disease affecting central nervous system. A potential therapeutic approach should possess several properties: Prevent immune system from damaging the brain and spinal cord, promote differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes to produce myelin, prevent the formation of fibronectin aggregates by astrocytes to inhibit scar formation, and enhance function of healthy endothelial cells (ECs).

Methods: To determine if an increase in sulfur contents through H2S, a potent antioxidant known to induce protective autophagy in cells, could provide the above desired outcomes, peripheral blood mononuclear cells (PBMNCs), OCPs, astrocytes, and ECs were treated with NaHS (50 μM) in vitro.

Results: Transmigration assay using EC monolayer showed that serotonin increased migration of PBMNC while pretreatment of EC with NaHS inhibited the migration induced by serotonin treatment. NaHS upregulated proteins involved in immune system response and downregulated PBMNCs- and EC-related adhesion molecules (LFA-1 and VCAM-1). Furthermore, it had a cell expansion inducing effect, altering EC morphology. The effects of NaHS on OPCs and astrocytes were studied compared to mTOR inhibitor rapamycin. In NaHS treated astrocytes the induced fibronectin production was partially inhibited while rapamycin almost fully inhibited fibronectin production. NaHS slowed but did not inhibit the differentiation of OCPs or the production of myelin compared to rapamycin.

Conclusion: The in vitro results point to the potential therapeutic application of hydrogen sulfide releasing molecules or health-promoting sulfur compounds in MS.

Keywords: Astrocytes; Fibronectin; HUVEC; Myelin; NaHS; Oligodendrocytes; Peripheral Blood Mononuclear Cells.

Figure 1.

Effect of serotonin on peripheral blood mononuclear cells (PBMNCs), A) Transmigration of PBMNCs subjected to different treatments across endothelial cells (EC), B) IL-10 expression by PBMNCs through NaHS treatment, C) SOCS3 (suppressor of cytokine signaling), 3) Expression by PBMNCs through NaHS treatment, D) CBS (cystathionine beta synthase) immunohistochemical analysis in untreated PBMNCs, E) CBS immunohistochemical analysis in serotonin treated PBMNCs (1 hour), F) CBS expression in PBMNCs at 15 minutes and 1 hour after serotonin treatment and after CBS knockout, G) Integrin LFA-1 expression where PBMNCs were subjected to different treatments. Data are presented as means±SD (n≥3 per group in 3 replicate experiments). * Difference to control cells. # Difference to CBS siRNA treated cells (significant difference between dotted bars). P<0.05. Western blot expression is normalized to ß-actin; lanes of Western blot insets are in the same order as in the X-axis.

Figure 2.

Immunohistochemical and Western blot analysis of fibronectin in astrocytes treated with NaHS and mTOR inhibitor rapamycin, percentage cell survival and total cell number, A) Control cells, B) Poly (I:C) (fibronectin production stimulating molecule), C) Rapamycin-treated cells, D) NaHS-treated cells, E) Western blot analysis of fibronectin expression through Poly (I:C) treatment and the effect of rapamycin and NaHS on fibronectin after Poly (I:C) treatment, F) Percentage of viable cells after NaHS and rapamycin treatment, G) Total cell number of treated astrocytes after 120 hours compared to controls. Data are presented as means±SD (n≥3 per group in 3 replicate experiments). * Difference to control cells, # Difference to poly (I:C) treated cells (significant difference between black bars). ## Difference to 120 hours control. P<0.05. Western blot expression is normalized to ß-actin; lanes of western blot insets are in the same order as in the X-axis.

Figure 3.

Differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes (OLGs) shown by immunohistochemical and Western blot analysis of MBP (myelin basic protein) in OLGs treated with NaHS and mTOR inhibitor rapamycin and percentage cell survival, A) OPC, B) Oligodendrocytes, C) NaHS treated OPC through NaHS differentiate at a lower rate into oligodendrocytes with lower number of branching, D) high inhibition OPC differentiation and OLG branching by rapamycin, E) The effect of NaHS treatment on MBP expression, F) The effect of rapamycin treatment on MBP expression, G) NaHS effect on decrease in MBP expression by OLGs but to a lesser degree compared to rapamycin, H) NaHS effect on lowering cell viability while rapamycin highly decreased cell viability. Data are presented as means±SD (n≥3 per group in 3 replicate experiments). * Difference to normal cells. # Difference to rapamycin treated cells (significant difference between black bars). P<0.05. Western blot expression is normalized to ß-actin; lanes of Western blot insets are in the same order as in the X-axis.

Figure 4.

NaHS effects on endothelial cell morphology and expression of anti-inflammatory mediators, A) Changes of cell morphology and relaxation/expansion of HUVECs by NaHS, B) triangular island form with preferentially extending motile processes (lamellipodia) from their corners, C) the square expanded/relaxed shape (Astier, 2008), D) within 30 minutes cells occupy more space acquiring a larger cell area, E) NaHS increases HO-1 levels compared to control cells, (F) VCAM-1 expression on HUVEC after 1 hour pretreatment with NaHS and 24 hours treatment with lipopolysaccharide (LPS 100 ng/ml), G) SOCS-3 expression through NaHS treatment. Data are presented as means±SD (n≥3 per group in 3 replicate experiments). * Difference to normal cells. # Difference to LPS treated cells (significant difference between black bars). P<0.05. Western blot expression is normalized to ß-actin; lanes of Western blot insets are in the same order as in the X-axis.