The Effect And Related Mechanism Of Ketorolac Tromethamine On Acute Fulminant Hepatitis

Background and objective:Acute fulminant hepatitis had no safe and effective treatment plan due to itsacute illness, quick developed complications and many features. In pathogenesis, theactivation of monocytes, T cells and macrophages as well as the release ofinflammatory cytokines, such as TNF-α, IL-1β, endotoxin was the important partcausing large areas of liver necrosis. In recent years, the experts found thatconcanavalin A (ConA) could be a good simulation of the process in acute severehepatitis that provided a good basis for our further study. Nuclear factor kappa B (NF-κB) is an important nuclear transcription factor related with the occurrence ofinflammation and development of tumors in vivo. Ketorolac tromethamine is acommonly used non-steroidal anti-inflammatory drug and studies showed that theantioxidantandante-inflammatory activities may be related to the inhibition of NF-κBpathway in vivo. This study referred to observe the protective effects of ketorolactromethamine on ConA-induced acute severe hepatitis in mice and explore thepreliminary mechanism on the expression of NF-κB.Methods:1. Ninety Balb/C male mice were weighed and then randomly divided into threegroups:The normal control group (NC group): an equal volume of saline to drug wasinjected.The ConA model group (ConA group): a concentration of20mg/kg ConA wasinjected via tail veinThe drug treatment group (Drug group): ketorolac tromethaine wasintraperitoneally injected1h before ConAinjected, dosage for2mg/kg2. The mice in various groups were extracted eyeball blood and killed at6h,12h and24h after ConA injection. The liver enzymes and inflammatory cytokines (TNFα,IL-1β, IL-6and IFN-γ) were detected in separated serum and the pathologicalobservation was by hematoxylin and eosin staining. The time point of12h provedmost severe was selected as the observation time to extract RNA and total protein todetermine the expression of Bcl-2, Bax, IkB-α and IkB-β from gene and proteinlevels.The further detection of relationship between NF-κB and endogenous apoptosiswas assessed to explore the protective mechanism of ketorolac tromethamine.Results:1. The ALT and AST of the model group increased significantly compared to thecontrol group while two indicators declined at three time points to some extent afterketorolac tromethamine treatment,12h at the peak (P<0.05); At6h, The mildcongestion, edema and few necrosis appeared in the model group developed to thelarge amounts of inflammation factors, large necrosis at12h. The statistics of theedema and necrosis area showed that the drug treatment group was belo the modelgroup (P<0.05); theinflammation factors, such as TNF-α, IL-6, IL-1βand IFN-γ,was detected to show different degrees of increase and statistics significance locatedcompared with the control group (P<0.05); TNF-α reduced most obviously at6h, andIL-6, IL-1β and IFN-γ had a significant effect at12h.2. The Bcl-2and Bax genes were detected by PCR and western blot techniques.At12h, the level of Bcl-2in the ConA group was lower obviously than the controlgroup, but after being given ketorolac tromethamine the expression of Bcl-2increased,the difference had significant statistics (P<0.05). The pro-apoptosis factor Bax wasobjected to the Bcl-2. The TNF-α, IL-6, IL-1β and IFN-γ was detected by westernblot and immunohistochemistry technologies to show those in model group weresignificantly increased. And after drug treatment, the release of inflammatorycytokines accompanied with the inflammation declined.As the key proteins of the NF-κB pathway, IκB-α and IκB-β were detected by PCR, western blot andimmunohistochemical staining. NF-κB in the model groups was significantlyimproved, gene and protein levels of NF-κB was a downward trend, expression levels was close to the normal control group. The expressions of IκB-α and IκB-β showedopposite trends to the NF-κB, gray band values between groups were statisticallydifferent (P<0.05).Conclusion:1.Ketorolac tromethamine could effectively reduce serum levels of ALT and ASTin ConA-induced liver injury and improved the pathological changes and release ofinflammatory cytokines in the liver.2. Ketorolac tromethamine could reduce the release of inflammatory cytokines(TNF-α、 IL-1β、 IL-6、 INF-γ) and the percentage of Bax/Bcl-2via theinhibition of NF-κB to reduce liver cell apoptosis.