| The liver is the largest digestive gland in the human digestive system as well as the most important organ of drug metabolism. It has the biotranformation effect on certain interal metabolites and various non-nutrients in vitro and vivo, such as drugs and toxins. During the process of using medicine, illness caused by the immune allergic reaction of metabolites and hepatotoxicity resulted from medicine as well as its metabolites is called drug-induced liver injury.Acetaminophen(also known as paracetamol, Acetaminophen, APAP), is the most widely used anti-inflammatory drugs currently, but excessive use of this drug can cause hepatotoxicity, which will damage the liver[1]. Acetaminophen has been extensively used in clinical practice, but there is no good solution to inhibit the liver toxicity properly. Adenosine(adenosine) as well as its receptor attracts increasingly much attention due to its complex biological effects and significant regulatory function in a variety of diseases [2]. As an endogenous purine nucleoside, adenosine has, primarily by means of combining with G protein-coupled adenosine receptor(adenosine receptors, ARs) as well as activate it, an important effect on the many physical systems and organizations. ARs widely exist in liver cells[3], play an essential role[4-5] in the pathogenesis of liver disease, but its effect in the mechanism of drug-induced liver injury induced by acetaminophen has not been reported. Chan[6] and other studies have shown that liver cells release more adenosine when stimulated by drugs such as methotrexate,and adenosine receptor though c AMP-PKA signalingpathway involve in engendering liver inflammation. What’s more according to Alchera[7] and other studies,the increase of adenosine leads to liver hypoxia, inflammation and acute injury, also excess acetaminophen can cause liver cell damage as well[8-9].It can be reasoned that during the process of the drug-induced liver injury caused by acetaminophen,acetaminophen led to the damage of liver cells by activating of ARs, which c AMP-PKA signaling pathway also involved in. This experiment aimed to explore the effects of ARs as well as the c AMP-PKA signaling pathway mediated by ARs on the drug-induced liver injury caused by acetaminophen though the drug-induced liver injury model made by intragastric administration with acetaminophen. The main contents can be summarized as follows:1 The establishment of model of acetaminophen-induced liver injuryFifty male Kunming mice were randomly divided into normal control group and model group. Model group was treated with paracetamol 500 mg/kg and control group was treated with the same of concentration normal saline by intragastric administration, respectively. Twenty mice were killed after 24 hours. Serum AST, ALT, ALP, TBA were measured. The histological analysis was performed by HE staining. The last mice were used for isolation of hepatocytes.2. Effects of adenosine receptor modulators overall model of drug-induced liver damage caused by acetaminophenOne hundred and sixty male Kunming mice were randomly divided into normal control group, model group, CPA group, DPCPX group, CPA+DPCPX group, CGS group, ZM241385 group, CGS+ZM241385 group. Control group was treated with the500 mg/kg normal saline and other groups were treated with paracetamol 500 mg/kg by intragastric administration, respectively. Then were given CPA group, DPCPX group, CPA + DPCPX group, CGS group, ZM241385 group, CGS group +ZM241385 group: CPA(0.05 mg / kg, ip), DPCPX(0.1 mg / kg), CPA + DPCPX, CGS21680(0.2 mg / kg, ip), ZM241385(0.1 mg / kg), CGS21680 + ZM241385. All mice were killed after 24 hours. Serum AST, ALT, ALP, TBA were measured. The histological analysis was performed by HE staining. The last mice were used for isolation of hepatocytes.3 Isolation, cultivation and identification of HepatocytesThe male Kunming mice(40-60g) were anaesthetized with 10% chloral hydrate(3 ml / kg) and disinfected their abdominal skin with alcohol. Open the abdominal cavity and push intestines to the right side in order to expose the portal vein. Cannulate the portal vein. Perfuse the liver with pre-warm EGTA solution and 0.075% I collagenase. Remove and place the liver in a tissue culture dish and cut liver with iris scissors then add D-hanks and perfusion medium into tissue culture dish. Shake the tube in a titer plate shaker at room temperature for 15 min. Discard supernatant, re-suspend cell pellets in culture medium with 25% fetal bovine serum DMEM.4 The expression level of ARs m RNA and protein in the model of paracetamol-induced hepatotoxicityAccording to the previous studies,drug-induced liver injury was most obvious after adding paracetamol of 500mg/kg for 24 h. Therefore, paracetamol of 500mg/kg was chosen to make the model of paracetamol-induced hepatotoxicity and we also set up a normal group. We used q RT-PCR and Western Blot to detect the expression levels of A1 R, A2 AR, A2 BR, A3 R. In terms of the result, the levels of A1 R, A2 AR, A2 BR and A3 R expressed in the model group as well as the control group. Compared with the normal group,the expression levels of A1 R and A2 AR in the model group are much higher.5 The effect of c AMP signal pathway mediated by adenosine A1 receptor in the model of paracetamol-induced hepatotoxicityThe hepatocytes were seeded in the plate and incubated for 24 hours.Then five groups were divided randomly in this experiment: the control group, the model group, the PTX group(Adenosine A1 receptor mediated Gi/o protein inhibitor), the NECA group(no-selective adenosine receptor agonist) and the combination group. We added paracetamol of 500mg/kg into each group for 24 h to establish the model of paracetamol-induced hepatotoxicity except the control group, then PTX was added into the PTX group and the combination group for 24 h, finally NECA was added into the NECA group and the combination group for 15 min. The c AMP levels were measured with a c AMP radio immunoassay kit in terms of the manufacturer’s instructions as well as the protein expression levels of PKA and p-CREB which were detected by Western Blot. Experiments were repeated for three times. According to the results,the expression of c AMP and the key signal molecules, like PKA and p-CREB were significantly increased when compared with the normal group. What’s more, PTX can obviously enhance the effect of NAEC. Based on the results, PTX as Gi/o protein coupled receptor inhibitor, can increase the production of c AMP by inhibiting Gi/o- AC- c AMP channel and enhance the effect of nonselective AR agonist.To further research on the effect of adenosine A1 R and A1R-mediated c AMP signaling pathway in the model of paracetamol-induced hepatotoxicity, respectively cultivate hepatocytes with adenosine A1 R agonist CPA, A1 R antagonist DPCPX, CPA and DPCPX for 48 hours. According to the further study,the levels of c AMP, PKA and p-CREB in the model group were significantly decreased after CPA treatment. And DPCPX can further increase the expression levels of c AMP, PKA andp-CREB in the model group. At the same time, the effect above in the combination group was obviously reversed. The results suggested that the A1R-Gi/o-AC-c AMP-PKA-CREB signaling pathway has a certain role within the the model of paracetamol-induced hepatotoxicity6 The effect of c AMP signal pathway mediated by adenosine A2 A receptor in the model of paracetamol-induced hepatotoxicityThe hepatocytes were seeded in the plate and incubated for 24 hours.Then five groups were divided randomly in this experiment: the control group, the model group, the Rolipram group, the NECA group and the NECA+Rolipram group. We added paracetamol of 500mg/kg into each group for 24 h to establish the model of paracetamol-induced hepatotoxicity except the control group, then Rolipram was added into the Rolipram group and the NECA+Rolipram group for 15 min, NECA was added into the NECA group and the NECA+Rolipram group for 10 min finally. The levels of c AMP were measured with a c AMP radio immunoassay kit in terms of the manufacturer’s instructions and the protein expression levels of PKA and p-CREB were detected by Western Blot. Experiments were repeated at for three times. According to the result, the expression of c AMP and the key signal molecules, like PKA and p-CREB were significantly increased when compared with the normal group. Rolipram and NECA can obviously increase the level of c AMP, PKA and p-CREB.in the model group. The combination of the Rolipram can and NECA can further enhance the effects. The level of c AMP can be increased in two ways: First, as the antagonists of ARs; Second, inhibition of the intracellular phosphodiesterase activity. The results indicate that Rolipram as phosphodiesterase inhibitors(IV) significantly increase level of c AMP, and NECA as non-selective AR agonist also significantly increase level of c AMP, prompt the presence of AR-Gs-AC-c AMP signaling pathways in hepatocytes.To further research on the role of adenosine A1 R and A1R-mediated c AMP signaling pathway in the model of paracetamol-induced hepatotoxicity, respectively cultivate hepatocytes with adenosine A2 AR agonist CGS21680, adenosine A2 AR antagonist ZM241385, or CGS21680+ ZM241385 for 48 hours. According to the further study,the protein expression levels of c AMP, PKA and p-CREB in the model group were obviously reduced after the ZM241385 treatment. However the levels were further increased after the treatment of CGS21680.Whereas the effect above in the ZM241385+CGS21680 combination group was significantly reversed. The result indicates that the A2AR-Gs-AC-c AMP-PKA-CREB signaling pathway has a certain role during the model of paracetamol-induced hepatotoxicity.In conclusion, the expression levels of adenosine A1 R and A2 AR in the model of paracetamol-induced hepatotoxicity are much higher than the normal hepatocytes. Adenosine A1 R and A2 AR jointly participate in regulating the model of paracetamol-induced hepatotoxicity.Even both of the A1 R and A2 AR antagonists have obvious inhibitory effect in the model of paracetamol-induced hepatotoxicity, they have opposite effects on c AMP-PKA-CREB signaling pathway. |
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