Extracellular vesicles from mesenchymal stem cells reverse neuroinflammation and cognitive and motor impairment in rats with mild liver damage

A high percentage of cirrhotic patients develop minimal hepatic encephalopathy (MHE), with mild cognitive impairment, psychomotor slowing, and motor incoordination. MHE reduces life quality and span of patients and increases the risk of suffering clinical hepatic encephalopathy (HE). Mild cognitive impairment in patients with no-alcoholic fatty liver disease (NAFLD) has been also recently reported. Human mesenchymal stem cells derived extracellular vesicles (MSC-EVs) have been proposed as a therapy for different diseases including liver injury and neurodegenerative diseases, due to their immunomodulatory, anti-inflammatory, and regenerative properties. The administration of carbon tetrachloride (CCl4) to rats is a model of progressive liver damage and HE. The main objective of this study was to assess if MSC-EVs reverse cognitive and motor impairment in rats with mild liver damage induced by CCl4 and MHE, as a model of NAFLD, and to expand the knowledge about the underlying mechanisms. In addition, we assessed if TGF-β, a main component of MSC-EVs and with anti-inflammatory properties, has a role in the effects of MSC-EVs on mild liver damage consequences. Mild liver damage was induced by intraperitoneal injection of CCl4 three times per week, for four weeks. MSC-EVs were isolated from human adipose tissue-derived stem cells and also from these cells after silencing TGF-β expression to assess the role of TGF-β. MSC-EVs were injected intravenously the second and third weeks of CCl4 treatment. Cognitive and motor functions were assessed. Motor coordination was analyzed in different behavioral tests including MotoRater, Beam walking, and Rotarod tests. Locomotor gait was assessed in the Catwalk system. Spatial memory was measured in the Novel Object Location test (NOL) while short-term memory was assessed in a Y-maze. Spatial learning index and working memory were measured in a radial maze. Glutamatergic and GABAergic neurotransmission and neuroinflammation in the cerebellum and the hippocampus were analyzed by immunohistochemistry and Western blot. Peripheral inflammation was measured with ELISA kits and western blot. Liver damage was determined by histopathological analysis in hematoxylin and eosin and Masson trichrome liver stains; and with the analysis of several markers of liver firbosis. Gut microbiota was also analyzed. MSC-EVs improve spatial learning and memory and working memory in rats with mild liver damage reducing neuroinflammation and normalizing some alterations in glutamatergic and GABAergic neurotransmission in the hippocampus. MSC-EVs improve motor coordination and altered locomotor gait by reducing neuroinflammation and the activation of pathways induced by TNFα that lead to GABAergic neurotransmission alterations. MSC-EVs also reduce peripheral inflammation, liver steatosis and fibrosis, and liver inflammation and counteract some consequences of gut dysbiosis. These effects are different depending on if the EVs proceed from unmodified MSCs or from MSCs with silenced TGF-β, indicating that this modification and the presence of TGF-β, that should also affect the rest of his cargo, is determinant to the effects of MSCs-EVs and the involved mechanisms. MSC-EVs could be a promising therapy for MHE and HE, and also for other diseases involving similar pathological mechanisms, such as diseases associated with sustained peripheral inflammation and neuroinflammation.

Ministerio de Universidades, Ayuda Formación de Profesorado Universitario FPU18/03851

Ministerio de Universidades, Ayudas a la movilidad para estancias breves y traslados temporales de beneficiarios FPU EST22/00527

Ministerio de Economía y Competitividad SAF2017-82917-R

Ministerio de Ciencia e Innovación PID2020-113388RB-I00

Generalitat Valenciana PROMETEU/2018/051; CIPROM/2021/082

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