The Influence of Caregiving on the Relation Between Marital Status and Psychological Health in a Grandparent Sample.

Grandparent caregivers report poorer psychological and physical health, but relationship status has been shown to influence burden. The current study investigated depressive symptoms of 3288 grandparents who completed the third wave of the National Survey of Families and Households. The study found that those who are unmarried were more likely to be grandparent caregivers, and female participants reported higher depressive symptoms. Marital status and caregiving status were comparable predictors of depression, but marital status did not buffer the effects of caregiving status on depression. Caregiving status accounted for a significant amount of depressive symptom variance for depression, comparable to marital status and gender. There was a significant difference in depressive symptoms of married and unmarried grandparent non-caregivers but with a significantly lower baseline depression rate than grandparent caregivers. Future research should examine whether making social support options available to unmarried grandparent caregivers who lack informal support from a spouse may improve outcomes.

Myostatin regulates the fibrogenic phenotype of hepatic stellate cells via c-jun N-terminal kinase activation.

BACKGROUND & AIMS: Myostatin is mainly expressed in skeletal muscle, where it negatively regulates trophism. This myokine is implicated in the pathophysiology of nonalcoholic steatohepatitis, an emerging cause of liver fibrosis. In this study we explored the effects of myostatin on the biology of hepatic stellate cells. METHODS: The effects of myostatin were assessed both in LX-2 and in human primary stellate cells. Cell migration was determined in Boyden chambers. Activation of intracellular pathways was evaluated by Western blotting. Procollagen type 1 secretion was measured by enzyme immunoassay. The role of c-Jun N-terminal kinase was assessed by pharmacologic and genetic inhibition. RESULTS: Activin receptor-2B was up-regulated in livers of mice with experimental fibrosis, and detectable in human stellate cells. Serum myostatin levels increased in a model of acute liver injury. Myostatin reduced HSC proliferation, induced cell migration, and increased expression of procollagen type1, tissue inhibitor of metalloproteinase-1, and transforming growth factor-ß1. Myostatin activated different signaling pathways, including c-Jun N-terminal kinase and Smad3. Genetic and/or pharmacologic inhibition of c-Jun N-terminal kinase activity significantly reduced cell migration and procollagen secretion in response to myostatin. CONCLUSIONS: Activation of activin receptor-2B by myostatin modulates the fibrogenic phenotype of human stellate cells, indicating that a myokine may be implicated in the pathogenesis of hepatic fibrosis.

NLRP3 inflammasome as a target of berberine in experimental murine liver injury: interference with P2X7 signalling.

Berberine (BRB) is commonly used in herbal medicine, but its mechanisms of action are poorly understood. In the present study, we tested BRB in steatohepatitis induced by a methionine- and choline-deficient (MCD) diet, in acute acetaminophen intoxication and in cultured murine macrophages. BRB markedly improved parameters of liver injury and necroinflammation induced by the MCD diet, although increased mortality was observed by mechanisms independent of bacterial infections or plasma levels of BRB. The MCD diet induced up-regulation of all components of the NLRP3 (NACHT, LRR and PYD domain-containing protein 3) inflammasome, and increased hepatic levels of mature IL-1ß (interleukin 1ß). All of these parameters were significantly reduced in mice treated with BRB. In mice administered an acetaminophen overdose, a model dependent on inflammasome activation, BRB reduced mortality and ALT (alanine aminotransferase) elevation, and limited the expression of inflammasome components. In vitro, LPS (lipopolysaccharide)-induced activation of NLRP3 inflammasome in RAW264.7 murine macrophages was markedly decreased by pre-incubation with BRB. BRB significantly limited the activation of the purinergic receptor P2X7, involved in the late phases of inflammasome activation. Upon P2X7 knockdown, the ability of BRB to block LPS-induced secretion of IL-1ß was lost. These data indicate that administration of BRB ameliorates inflammation and injury in two unrelated murine models of liver damage. We demonstrate for the first time that BRB interferes with activation of the NLRP3 inflammasome pathway in vivo and in vitro, through a mechanism based on interference with activation of P2X7, a purinergic receptor involved in inflammasome activation.

Hepatic Notch1 deletion predisposes to diabetes and steatosis via glucose-6-phosphatase and perilipin-5 upregulation.

Notch signaling pathways have recently been implicated in the pathogenesis of metabolic diseases. However, the role of hepatic Notch signaling in glucose and lipid metabolism remains unclear and needs further investigation as it might be a candidate therapeutic target in metabolic diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). We used hepatocyte-specific Notch1 knockout (KO) mice and liver biopsies from NASH and NAFLD patients to analyze the role of Notch1 in glucose and lipid metabolism. Hepatocyte-specific Notch1 KO mice were fed with a high fat diet (HFD) or a regular diet (RD). We assessed the metabolic phenotype, glucose and insulin tolerance tests, and liver histology. Hepatic mRNA expression was profiled by Affymetrix Mouse Gene arrays and validated by quantitative reverse transcription PCR (qPCR). Akt phosphorylation was visualized by immunoblotting. Gene expression was analyzed in liver biopsies from NASH, NAFLD, and control patients by qPCR. We found that Notch1 KO mice had elevated fasting glucose. Gene expression analysis showed an upregulation of glucose-6-phosphatase, involved in the final step of gluconeogenesis and glucose release from glycogenolysis, and perilipin-5, a regulator of hepatic lipid accumulation. When fed with an HFD KO mice developed overt diabetes and hepatic steatosis. Akt was highly phosphorylated in KO animals and the Foxo1 target gene expression was altered. Accordingly, a reduction in Notch1 and increase in glucose-6-phosphatase and perilipin-5 expression was observed in liver biopsies from NAFLD/NASH compared with controls. Notch1 is a regulator of hepatic glucose and lipid homeostasis. Hepatic impairment of Notch1 expression may be involved in the pathogenesis of human NAFLD/NASH.

n-3 polyunsaturated fatty acids worsen inflammation and fibrosis in experimental nonalcoholic steatohepatitis.

BACKGROUND & AIMS: n-3 polyunsaturated fatty acids (PUFA) ameliorate fatty liver in experimental models, but their effects on inflammation and fibrosis during steatohepatitis are either controversial or lacking. We compared the effects of supplementation with olive oil (OO) alone or OO and n-3 PUFA on the development and progression of experimental steatohepatitis. METHODS: Balb/C mice (≥5 mice/group) were fed a methionine- and choline-deficient (MCD) diet or a control diet for 4 or 8 weeks. At the same time, mice were supplemented with n-3 PUFA (eicosapentaenoic and docosahexahenoic acid, 25 mg together with 75 mg OO), or OO alone (100 mg), two times a week by intragastric gavage. RESULTS: After 8 weeks, mice on MCD/n-3 had higher ALT levels compared to MCD/OO and more severe scores of inflammation, including a significant increase in the number of lipogranulomas (26.4 ± 8.4 vs. 5.1 ± 5 per field, P < 0.001). Intrahepatic expression of TNF-α and CCL2 was higher in MCD/n-3 mice at both time points. In addition, increased expression of the profibrogenic genes TIMP-1 and TGF-ß, and more severe histological scores of fibrosis were evident in MCD/n-3 mice. After 8 week of MCD diet, portal pressure was higher in mice receiving n-3 than in those on OO alone (5.1 ± 1.4 vs. 7.0 ± 0.9 mmHg, P < 0.05). Analysis of hepatic fatty acid profile showed that supplementation resulted in effective incorporation of n-3 PUFA. CONCLUSIONS: In a murine model of steatohepatitis, supplementation with n-3 PUFA and OO is associated with more severe necro-inflammation and fibrosis than in mice treated with OO only.

Lack of CC chemokine ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis.

Expression of CCL2 (CC chemokine ligand 2) (or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favouring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In the present study, we investigated the development of non-alcoholic steatohepatitis induced by an MCD diet (methionine/choline-deficient diet) in mice lacking the CCL2 gene on two different genetic backgrounds, namely Balb/C and C57/Bl6J. WT (wild-type) and CCL2-KO (knockout) mice were fed on a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice fed on the MCD diet, a lack of CCL2 was associated with lower ALT (alanine transaminase) levels and reduced infiltration of inflammatory cells, together with a lower generation of oxidative-stress-related products. Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice. This was associated with reduced hepatic expression of TGF-ß (transforming growth factor-ß), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing WT and CCL2-KO animals fed on an MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL (interleukin)-4 and the M2 marker MGL-1 (macrophage galactose-type C-type lectin 1) were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice fed on the MCD diet. We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

Modulation of liver fibrosis by adipokines.

Hepatic fibrosis is an integrated process triggered by chronic liver damage, leading to the accumulation of extracellular matrix. In patients with chronic liver disease, this process is favored by the presence of obesity or overweight, which are also relevant risk factors for the progression of nonalcoholic steatohepatitis. In this paper, we review the available evidence indicating the modulation of the fibrogenic process by adipokines, a group of cytokines secreted primarily by adipose tissue. In particular, we discuss in detail the role of leptin and adiponectin, which favor and limit the fibrogenic process, respectively. The possible involvement of other recently identified adipokines is also briefly outlined.

Curcumin limits the fibrogenic evolution of experimental steatohepatitis.

Nonalcoholic steatohepatitis is characterized by the association of steatosis with hepatic cell injury, lobular inflammation and fibrosis. Curcumin is known for its antioxidant, anti-inflammatory and antifibrotic properties. The aim of this study was to test whether the administration of curcumin limits fibrogenic evolution in a murine model of nonalcoholic steatohepatitis. Male C57BL/6 mice were divided into four groups and fed a diet deficient in methionine and choline (MCD) or the same diet supplemented with methionine and choline for as long as 10 weeks. Curcumin (25 microg per mouse) or its vehicle (DMSO) was administered intraperitoneally every other day. Fibrosis was assessed by Sirius red staining and histomorphometry. Intrahepatic gene expression was measured by quantitative PCR. Hepatic oxidative stress was evaluated by staining for 8-OH deoxyguanosine. Myofibroblastic hepatic stellate cells (HSCs) were isolated from normal human liver tissue. The increase in serum ALT caused by the MCD diet was significantly reduced by curcumin after 4 weeks. Administration of the MCD diet was associated with histological steatosis and necro-inflammation, and this latter was significantly reduced in mice receiving curcumin. Curcumin also inhibited the generation of hepatic oxidative stress. Fibrosis was evident after 8 or 10 weeks of MCD diet and was also significantly reduced by curcumin. Curcumin decreased the intrahepatic gene expression of monocyte chemoattractant protein-1, CD11b, procollagen type I and tissue inhibitor of metalloprotease (TIMP)-1, together with protein levels of alpha-smooth muscle-actin, a marker of fibrogenic cells. In addition, curcumin reduced the generation of reactive oxygen species in cultured HSCs and inhibited the secretion of TIMP-1 both in basal conditions and after the induction of oxidative stress. In conclusion, curcumin administration effectively limits the development and progression of fibrosis in mice with experimental steatohepatitis, and reduces TIMP-1 secretion and oxidative stress in cultured stellate cells.

Mononuclear cells in liver fibrosis.

Fibrosis is a multicellular wound healing process, where myofibroblasts that express extracellular matrix components extensively cross-talk with other cells resident in the liver or recruited from the bloodstream. Macrophages and infiltrating monocytes participate in the development of fibrosis via several mechanisms, including secretion of cytokines and generation of oxidative stress-related products. However, macrophages are also pivotal in the process of fibrosis resolution, where they contribute to matrix degradation. T lymphocytes modulate the fibrogenic process by direct interaction with myofibroblasts and secreting cytokines. In general, Th2 polarized responses promote fibrosis, while Th1 cytokines may be antifibrogenic. NK cells limit the development of fibrosis and favor its resolution, at least in part via killing of fibrogenic cells. The possible role of NKT cells and B cells is emerging in recent studies. Thus, mononuclear cells represent a critical regulatory system during fibrogenesis and may become an appealing target for therapy.