Expression Of Inflammasome-associated Molecules In Acute Rejection Of Liver Transplantation

Liver transplantation has been one of the most effective methods to treat end-stage liver diseases. The rejection after liver transplantation is still an intractable problem to be treated. Inflammation is the main manifestation of rejection. The proinflammatory cytokines secreted by inflammatory cells such as Interleukin(IL)-1, IL-2, IL-6, IL-10, Interferon-γ(IFN-γ) play an important role in rejection by increasing the permeability of vessels, enhancing the adhesion of leukocytes, destructing the normal tissues by infiltrated inflammatory cells and so on.Inflammasome is a cellular muti-molecule complex, containing NOD-like receptor, adaptor ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) and caspase-1. Inflammasome could activate caspase-1, which processes pro-IL-1β and pro-IL-18, resulting in the secretion of IL-1β and IL-18and having effects in regulating inflammatory responses and other biological reactions.The accumulating reports in recent years have demonstrated the important role of inflammasome in autoimmune diseases, such as chronic arthritis, systemic lupus eythematosus. The involvement of inflammasome in organ transplantation rejection has been also payed much attention.The genotypes of genes affiliated to inflammasome are pratical index of clinical outcomes after stem cell transplantation. The acute rejection of murine heart transplantation is closely related to inflammasome activating pathway. Hepatic ischemia/reperfusion injury is also correlated with inflammasome activation.In the present study, we use rat orthotopic liver transplantation (OLT) model to evaluate the relationship between expression of inflammasome-associated molecules and liver transplantation rejection and its mechanism, also to provide experimental and theoretical basis for preventing rejection. Three groups were divided. Lewis livers were transplanted into the same strain as isografts (Lewis-Lewis), DA livers were transplanted into Lewis recipients as allografts (DA-Lewis), and recipient of allogenetic OLT treated with Ac-YVAD-CMK, the inreversible inhibitor of caspase-1, through intraperitoneal injection(10mg/kg) each day after OLT(DA-Lewis treated with Ac-YVAD-CMK) as another group in the establishing45models of rat OLT by the " two sleeve method". Samples were collected on Dayl, Day3, Day7after OLT (n=5). H&E histopathologic analysis and Banff criteria were used to observe the degree of rejection. The protein expression of ASC and IL-1β in the grafts was detected by immunohistochemical analysis. The-relative levels of ASC, IL-1β and IFN-γ mRNA were assessed by real time reverse transcript-polymerase chain reaction (RT-PCR). IL-1β protein in serum was detected by ELISA.The results showed:(1) Compared with isografts, the inflammation reaction in allografts was intensified in parallel to the time after transplantation.The inflammatory cells gathered around vessels and infiltrated into the hepatic parenchyma, hepatic cells necrosis ememerged obviously. The inflammation reaction in allografts treated with Ac-YVAD-CMK was lessened. RAI (rejection-active index) of isografts was significantly decreased (p<0.01) compared with allografts during acute rejection of liver transplantation course and remained low scores according to Banff criteria.. The RAI of allografts increased in a time-dependent manner after transplantation (p<0.01). Conversely, the RAI of Ac-YVAD-CMK-treated allografts was reduced similar to isografts. The result indicated that the inhibition of inflammasome activation could attenuate the liver transplantation rejection.(2) Compared with isografts, the expression of ASC and IL-1β in allografts investigated immunohistochemically was significantly increased with the development of rejection, showing apparent correlation. ASC was mainly located in the cytoplasm of inflammatory cells and IL-1(3was distributed in the interspace of hepatocytes around the vessels. In Ac-YVAD-CMK-treated allografts the expression pattern was similar to isografts.(3) The expression of ASC and IL-1β mRNA assessed by real time RT-PCR in allografts significantly elevated compared with isografts (p<0.05) and had correlation. In Ac-YVAD-CMK-treated allografts the expression of ASC and IL-1β mRNA was alike to isografts. Expression of IFN-y mRNA in untreated allografts was maximal on Day3while Ac-YVAD-CMK-treated allgrafts and isografts remained at the low level during the time course.(4)The ELISA result showed IL-1β in serum increased significantly on Day7after OLT campared with isografts(p<0.05). There was no significantly difference between isografts and Ac-YVAD-CMK-treated allografts.In conclusion, the upregulation of ASC in grafts could induce the productin of IL-1β, mediating inflammation reaction. Caspase-1inhibitor Ac-YVAD-CMK could effectively reduce the production of IL-1β through blocking caspase-1and attenuate the inflammation of rejection. The inflammasome activation pathway may be involved in pathology of rejection after liver transplantation. Therefore, the results provide the experimental and theoretical basis to explore the mechanism of liver transplantation rejection, also open up a noval preventive and therapyeutic approach for acute rejection of liver transplantation.