Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase

Abstract

The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers α-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers α-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O2(-)) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, α-SMA, collagen I, and TIMP-1 by inhibition of O2(-) production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O2(-) production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.

Activation of NLRP3 inflammasomes in mouse hepatic stellate cells during Schistosoma J. infection

“…Since our previous studies have shown that SEA may increase NADPH oxidase activity and enhanced production of ROS from activated NADPH oxidase activate inflammasomes [4, 28], the present study focused on how SEA acts through altered lysosome permeability and cathepsin B activity. We demonstrated that silencing of cathepsin B gene markedly inhibited SEA-induced co-localization of NLRP3 with ASC or caspase-1 (Figure 6A and 6B) and reduced SEA-enhanced cleavage of pro-caspase-1 (Figure 6C and 6D), caspase-1 activity (Figure 6E) and production of IL-1β (Figure 6F).…”

“…This results in a rapid assembling of NLRP3 inflammasome activating caspase-1 to produce IL-1β and other pathogenic factors to trigger inflammatory response [36–41] or directly promote fibrogenesis in some types of cells such as HSCs. In a recent study, we have demonstrated that Schistosoma J. infection or SEA activated NADPH oxidase (NOX) to produce O 2 .− and inhibition NOX activity or silencing NOX subunit gene blocked SEA-induced O 2 .− production and prevented liver fibrosis and related fibrogenic changes in HSCs [4]. Taken together, these results establish a new working model to induce SSLF, where Schistosoma J. infection or SEA activate NOX in HSCs to produce ROS and thereby activate NLRP3 inflammasomes, leading to caspase-1 activation and consequent production of different factors that instigate the local inflammatory response or induce fibrogenic changes in the liver.…”

“…Mouse HSCs were prepared by the discontinuous density gradient centrifugation technique as previously described [47] and some minor modifications were made to increase success rate as we described previously [4, 31]. The collected cells were cultured in DMEM (Gibco, Carlsbad, CA) containing 10% FBS (Gibco) in humidified 95% air and 5% CO2 mixture at 37°C.…”

“…Over 249 million individuals in 73 countries around the world are infected with different types of schistosoma, and nearly 800 million people are under the threat of such infection [1–4]. Although various aetiological factors may lead to chronic liver injuries toward hepatic fibrosis or even cirrhosis such as drug abuses, alcoholism, hepatitis B and C viral infection, Schistosoma J. infection is one of the most common causes for hepatic fibrosis in some countries [5, 6].…”

Immunoregulatory Role of Cannabinoids during Infectious Disease

“…This mechanism is partly due the CB1 activation in hepatic 195 stellate cells and depending on O 2 -production by NAPDH oxidase, and suggests the blockage of CB1 being employed as a therapeutic alternative in the prevention and treatment of hepatic cirrhosis or infection with Schistosoma [103].…”