ZT-1a

STK39 is reportedly a critical negative regulator of intestinal barrier. Pharmacological targeting of STK39 is expected to protect the intestinal barrier and thereby weaken metabolic dysfunction-associated steatohepatitis (MASH); Proximal colon biopsy tissues from patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and those without MASLD were analyzed for STK39 expression. Wildtype (WT) mice and systemic STK39 gene knockout (STK39^-/-) male mice were fed a normal diet or a high-fat methionine-choline deficient diet (HFMCD) for 8 weeks. The MASH mice were grouped and treated with ZT-1a (a STK39 inhibitor) or vehicle intraperitoneal injection during the procedure of HFMCD induction. Liver and intestinal tissues were collected for further examination; Colon tissues from patients with MASLD exhibited higher levels of STK39 than those from subjects without MASLD. Knockout of STK39 diminished CD68⁺ Kupffer cells and α-SMA⁺ hepatic stellate cells infiltration in mouse MASH model. Treatment with ZT-1a also prevented severe steatohepatitis in a mouse MASH model, including milder histological and pathological manifestations (lobular inflammation and fibrosis) in the liver. Interestingly, Inhibition of STK39 had minimal effects on hepatic lipid metabolism. The reduced liver injury observed in mice with STK39 inhibition was linked to significant decreases in mucosal inflammation, tight junction disruption and intestinal epithelial permeability to bacterial endotoxins; Collectively, we have revealed that inhibiting STK39 prevents the progression of MASH by protecting the intestinal epithelial barrier.

Astrogliosis and white matter lesions (WML) are key characteristics of vascular contributions to cognitive impairment and dementia (VCID). However, the molecular mechanisms underlying VCID remain poorly understood. Stimulation of Na‐K‐Cl cotransport 1 (NKCC1) and its upstream kinases WNK (with no lysine) and SPAK (the STE20/SPS1‐related proline/alanine‐rich kinase) play a role in astrocytic intracellular Na+ overload, hypertrophy, and swelling. Therefore, in this study, we assessed the effect of SPAK inhibitor ZT‐1a on pathogenesis and cognitive function in a mouse model of VCID induced by bilateral carotid artery stenosis (BCAS).

Following sham or BCAS surgery, mice were randomly assigned to receive either vehicle (DMSO) or SPAK inhibitor ZT‐1a treatment regimen (days 14–35 post‐surgery). Mice were then evaluated for cognitive functions by Morris water maze, WML by ex vivo MRI‐DTI analysis, and astrogliosis/demyelination by immunofluorescence and immunoblotting.

Compared to sham control mice, BCAS‐Veh mice exhibited chronic cerebral hypoperfusion and memory impairments, accompanied by significant MRI DTI‐detected WML and oligodendrocyte (OL) death. Increased activation of WNK‐SPAK‐NKCC1‐signaling proteins was detected in white matter tissues and in C3d+GFAP+ cytotoxic astrocytes but not in S100A10+GFAP+ homeostatic astrocytes in BCAS‐Veh mice. In contrast, ZT‐1a‐treated BCAS mice displayed reduced expression and phosphorylation of NKCC1, decreased astrogliosis, OL death, and WML, along with improved memory functions.

BCAS‐induced upregulation of WNK‐SPAK‐NKCC1 signaling contributes to white matter‐reactive astrogliosis, OL death, and memory impairment. Pharmacological inhibition of the SPAK activity has therapeutic potential for alleviating pathogenesis and memory impairment in VCID.