Células madre adiposas humanas disminuyen el daño de la fibrosis hepática con baja persistencia de células trasplantadas en ratas / Human adipose stem cells decrease liver fibrosis damage with low persistence of transplanted cells in rats

RESUMEN: En la enfermedad hepática crónica el trasplante ortotópico es la única alternativa terapéutica actual pero es limitada por falta de donantes. Ensayos con células madre adultas en daño hepático agudo evidencian promisorios resultados. El objetivo de este trabajo fue evaluar en ratas con daño hepático crónico la efectividad de la infusión de células madre adiposas humanas (CMAd-h). Ratas con fibrosis hepática inducida por tioacetamida fueron agrupadas en: grupo I control que no recibió tioacetamida ni células madre, grupo II recibió tioacetamida y suero fisiológico i.v., grupo III recibió tioacetamida y células madre adiposas 1 x 106/kg i.v. vía vena de la cola. La regeneración hepática histológica se evaluó por el index METAVIR, mientras las Macrophagocytus stellatus, células estrelladas a- SMA+ y células colágeno I+ por inmunohistoquímica; el daño funcional se evaluó por los niveles sanguíneos de los analitos Aspartato Aminotransferasa (AST), Alanina Aminotransferasa (ALT), Fosfatasa Alcalina (ALP), úrea y nitrógeno ureico (BUN) y hemograma. Los resultados muestran atenuación del daño estructural hepático evidenciado por disminución de los nódulos, del grado de lesión histológica en el score Metavir, y disminución de Macrophagocytus stellatus, células a-SMA+ y células colágeno tipo I+; funcionalmente hay reducción moderada de AST, ALT, urea, BUN y disminución moderada de células blancas pero efecto favorable sobre el volumen corpuscular media y la hemoglobina corpuscular media. Ocho semanas después de la infusión hay escasa población de CMAd-h en el hígado. En conclusión la infusión intravenosa de CMAd-h en ratas disminuye el daño funcional y estructural de la fibrosis hepática con escasa persistencia de CMAd-h en el parénquima hepático. A nuestro conocimiento este es el primer trabajo que evalúa el efecto de las CMAd-h en el modelo daño hepático crónico murino y la persistencia de las células trasplantadas.

SUMMARY: In chronic liver disease, orthotopic transplantation is the only current therapeutic alternative but it is limited due to lack of donors. Trials with adult stem cells in acute liver damage show promising results. The aim of this work was to evaluate the effectiveness of human adipose stem cell (h-ASC) infusion in rats with chronic liver damage. Rats with thioacetamide- induced liver fibrosis were grouped into: group I control that did not receive thioacetamide and h-ASC, group II received thioacetamide and saline i.v., group III received thioacetamide and h-ASC 1 x 106/ kg i.v. via tail vein. Histological liver regeneration was evaluated by METAVIR index, while Macrophagocytus stellatus (Kupffer cells), stellate cells a-SMA+ and collagen I+ cells by immunohistochemistry; functional damage was evaluated by blood levels of the analytes Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP), Urea and Blood Urea Nitrogen (BUN) and hemogram. The results show attenuation of structural liver damage evidenced by decreased nodules, degree of histologic injury on Metavir score, and decreased Macrophagocytus stellatus, a-SMA+ cells and type I+ collagen cells; functionally there is moderate reduction of AST, ALT, urea, BUN and moderate decrease of white cells but favorable effect on mean corpuscular volume and mean corpuscular hemoglobin. Eight weeks after infusion there is a small population of h-ASC in the liver. In conclusion, intravenous infusion of h-ASC in rats reduces functional and structural damage of hepatic fibrosis with low persistence of h- ASC in the liver parenchyma. To our knowledge this is the first work that evaluates the effect of h-SC in the model of chronic murine liver damage and the persistence of transplanted cells.

Hepatotrophic factors reduce hepatic fibrosis in rats / Fatores hepatotróficos reduzem a fibrose hepática em ratos

CONTEXT: Hepatic fibrosis occurs in response to several aggressive agents and is a predisposing factor in cirrhosis. Hepatotrophic factors were shown to stimulate liver growth and to restore the histological architecture of the liver. They also cause an improvement in liver function and accelerate the reversion of fibrosis before it progresses to cirrhosis. OBJECTIVE: To test the effects of hepatic fibrosis solution composed by amino acids, vitamins, glucose, insulin, glucagon and triiodothyronine on hepatic fibrosis in rats. METHODS: Fibrosis was induced in rats by gastric administration of dimethylnitrosamine (10 mg/kg) for 5 weeks. After liver biopsy, the rats received either hepatotrophic factors solution (40 mg/kg/day) or saline solution for 10 days by intraperitoneal injection. Blood samples and liver fragments were collected for hepatic function analysis, standard histopathology evaluation, and morphometric collagen quantification. RESULTS: Rats in the hepatotrophic factors group showed a decrease of the histopathological components of fibrosis and an increase of their hepatic mass (12.2 percent). There was no development of neoplasic lesions in both groups. Compared with the saline group, the hepatotrophic factors group also had a decrease of blood levels of hepatic-lesion markers (AST, ALT) and a decrease of collagen content in the portal spaces (31.6 percent) and perisinusoidal spaces (42.3 percent), as well as around the hepatic terminal vein (57.7 percent). Thus, hepatotrophic factors administration in the portal blood promoted a regenerative hepatic response, with an overall reduction of the volumetric density of collagen, improved hepatic function, and a general improvement in the histopathological aspects of fibrosis. CONCLUSION: Taken together, these results suggest the potential therapeutic use of this hepatotrophic factors solution to treat chronic liver diseases.

CONTEXTO: A fibrose hepática ocorre em resposta a diversos agentes agressores e é um fator predisponente da cirrose. Fatores hepatotróficos são conhecidos por estimular o crescimento hepático e restaurar a arquitetura histológica do fígado. Promovem, também, melhora na função hepática e aceleram a reversão da fibrose antes de sua progressão para cirrose. OBJETIVO: Testar os efeitos de uma solução de fatores hepatotróficos, composta por aminoácidos, vitaminas, glicose, insulina, glugacon e triiodotironina na fibrose hepática em ratos. MéTODOS: No presente estudo, a fibrose foi induzida em ratos pela administração de dimetilnitrosamina (10 mg/kg) durante 5 semanas. Após a biopsia do fígado, os ratos receberam a solução de fatores hepatotróficos (40 mg/kg/dia) ou solução salina por injeção intraperitonial, durante 10 dias. Amostras sanguíneas e fragmentos do fígado foram coletados para análise da função hepática, avaliação do critério histopatológico e quantificação morfométrica do colágeno. RESULTADOS: Os ratos do grupo fatores hepatotróficos demonstraram diminuição dos componentes histopatológicos da fibrose e aumento de massa hepática (12,2 por cento). Não houve o desenvolvimento de lesões neoplásicas em ambos os grupos. Comparado com o grupo de salina, no grupo fatores hepatotróficos também houve diminuição nos níveis dos marcadores sanguíneos de lesão hepática (AST e ALT), e diminuição da quantidade de colágeno nos espaços porta (31,6 por cento) e espaços perissinusoidais (42,3 por cento), assim como ao redor das veias terminais hepáticas (57,7 por cento). Assim, a administração de fatores hepatotróficos no sangue portal promoveu resposta regenerativa hepática, com redução da densidade volumétrica de colágeno, melhora na função hepática e melhora geral nos aspectos histopatológicos da fibrose. CONCLUSÃO: Juntos, estes resultados sugerem o potencial uso terapêutico desta solução de fatores hepatotróficos para tratar doenças hepáticas crônicas.

Modulation of extracellular matrix by nutritional hepatotrophic factors in thioacetamide-induced liver cirrhosis in the rat

Nutritional substances associated to some hormones enhance liver regeneration when injected intraperitoneally, being denominated hepatotrophic factors (HF). Here we verified if a solution of HF (glucose, vitamins, salts, amino acids, glucagon, insulin, and triiodothyronine) can revert liver cirrhosis and how some extracellular matrices are affected. Cirrhosis was induced for 14 weeks in 45 female Wistar rats (200 mg) by intraperitoneal injections of thioacetamide (200 mg/kg). Twenty-five rats received intraperitoneal HF twice a day for 10 days (40 mL·kg-1·day-1) and 20 rats received physiological saline. Fifteen rats were used as control. The HF applied to cirrhotic rats significantly: a) reduced the relative mRNA expression of the genes: Col-α1 (-53 percent), TIMP-1 (-31.7 percent), TGF-β1 (-57.7 percent), and MMP-2 (-41.6 percent), whereas Plau mRNA remained unchanged; b) reduced GGT (-43.1 percent), ALT (-17.6 percent), and AST (-12.2 percent) serum levels; c) increased liver weight (11.3 percent), and reduced liver collagen (-37.1 percent), regenerative nodules size (-22.1 percent), and fibrous septum thickness. Progranulin protein (immunohistochemistry) and mRNA (in situ hybridization) were found in fibrous septa and areas of bile duct proliferation in cirrhotic livers. Concluding, HF improved the histology and serum biochemistry of liver cirrhosis, with an important reduction of interstitial collagen and increased extracelullar matrix degradation by reducing profibrotic gene expression.

Electrophoretic and histological studies on hepatic schistosomiasis and its treatment with certain drugs

Silymarin has been shown to have a hepatoprotective effect in the treatment of liver cirrhosis, fatty liver, acute and chronic hepatitis. Also pentoxifylline has antifibrosis effect and could improve the regeneration and function of the liver in mice. So, the aim of this study was to evaluate the influence of both silymarin and pentoxifylline as antfibrotic drugs with praziquantel or mirazid as antibilharzial drugs on mice infected with cercariae of Schistosoma mansoni. The experimental animals were classified into 4 groups each divided into 3 subgroups. All groups were sacrificed at 18[th] week after infection. The obtained results showed that the liver sections of animals treated with praziquantel demonstrated less histopathological changes and granulomatous lesions than those treated with mirazid. Also, praziquantel and silymarin as antifibrotic drug showed more effective protection than praziquantel and pentoxifylline. The same results were obtained with treatment with the drug mirazid. Few scattered small sized granulomatous lesions were present in mice liver sections treated with praziquantel with either silymarin or pentoxifylline than in case of mirazid with silymarin or pentoxifylline as antifibrotics. Moreover, no worms appeared in the liver sections of mice treated with either praziquantel drug or combined drugs with antifibrotics. The electrophoresis study showed that the changes in the total proteins and differential proteins [Gamma, Beta, Alpha, Albumin, Pre-albumin] were highly significant in infected animal group than in uninfected group. Also, there was highly significant change in differential proteins in the groups treated with combined drugs than in groups treated with one drug without antifibrotics which gave more protection in the treatment of schistosomiasis. The results also indicated that praziquantel and silymarin were more effective in the treatment of schistosomiasis than mirazid