Establishment Of A New Method For Dipeptidyl Peptidase-? Detection And Its Application In The Comparative Study In The Evaluation Of Organ Injury In Rats

Organ injury and specific evaluation of organ injury is the most basic and important task for diagnosis and evaluation in the clinic.Taking drug-induced organ injury as an example,timely detection of adverse reactions and toxic side effects of drugs are crucial for both the development of new drugs and clinical diagnosis and treatment.Since the drug was often used after diagnosis,identifying the cause and effect of the injury or the site of the original injury becomes a great challenge.Drug-induced organ damage,as a kind of exogenous damage,is difficult to distinguish from other pathogenic factors.Moreover,drug-induced organ injury also showed obvious inflammatory injury characteristics such as“DAMPs”(Damage Associated Molecular Patterns).Taking drug-induced liver injury as an example,there are usually three types of damage:1.Direct injury to tissue cells caused by heterologous substances;2.Accumulation of endogenous metabolites after direct injury,such as bile acid,bilirubin,which become the key events for secondary cell injury;3.the dysregulation and activation of the immune system,which was induced by endogenous substances released by injured cells or exogenous factors.Therefore,the organ injury might be caused by multiple factors.Due to the mixed mechanism and mode of DAMPs,liver injury presents two mainThe evaluation of organ specific characteristics of drug-induced organ injury includes histopathological diagnosis,which is determined by tissue and regarded as the gold standard of diagnosis,and detection of serum biomarker released from injured cells,which is used to indirectly evaluation the extent of injury.Therefore,several questions such as the tissue source of biomarkers,the abundance of biomarkers in the tissue and the residual activity of biomarkers in injured tissue need to be verified.The gold standard of histopathological diagnosis still faces many challenges.As a qualitative and descriptive analysis method,it has some limitations.For example,sample collection,doctors'experience or preference in diagnosis might affect the diagnostic results.Injury biomarkers have been extensively used in clinical diagnosis due to their advantages of simplicity,high selectivity and quantification.However,different markers have different characteristics.Due to the different relative distribution and abundance of markers in different individuals,organs and cells,efficiency and criteria of diagnostic methods are also different.Drug induced injury often has multiple characteristics and multiple mechanisms.Whether a certain marker in serum is derived from a certain injured tissue should be verified by using other markers with high tissue abundance.We intend to establish a new analytical method by using the combination of biomarkers outside liver with liver-specific markers as well as histomorphology examination,which would overcome the disadvantage of histomorphology in quantitative analysis.Therefore,comprehensive diagnostic performance of the current"gold standard"can be improved on the whole.Dipeptidyl peptidase-?(DPP-?)is a peptide hydrolase widely distributed and present in many tissues except liver.It specifically hydrolyzes alanine or proline located in the second position of the N terminal of the peptide chain.A variety of active peptides in vivo is activated or inactivated after DPP-? hydrolysis.Therefore,DPP-? has a variety of pathophysiological mechanisms in vivo,participates in a variety of metabolic processes,and is an important regulatory role.DPP-? has been extensively studied as a drug target,and is also a target for various cancer therapies in addition to type 2 diabetes.DPP-? is highly expressed in the kidney and intestine,and is also expressed in the liver,pancreas,placenta,thymus,etc.DPP-? also exists in the circulating blood in soluble form.Soluble DPP-?,which has the same hydrolase activity,is a potential marker for organ injury and has received extensive attention.Therefore,quantitatively detection of the DPP-? activityIn view of the above problems,the resulting work is carried out in this study:1.The primary task is to establish a DPP-? detection method with high specificity and sensitivity,to detect the specific activity of DPP-? in different tissues under Ctrl circumstances,and to use it as an effective marker to observe the heterogeneity of Ctrl tissues;2.To establish a method for the determination of tissue residual activity of DPP-? and make comparison with the changes of histopathological score,blood DPP-? level and other markers to analyze and confirm the source of the markers in blood or serum;3.Two recognized liver specific chemical damage agents were used to cause different liver injury in rats.The differences between two model was examined including histology,tissue residual activity and serum activity of traditional liver injury markers.4.Comparing the heterogeneity changes of DPP-? activity between different liver lobes or parts after the modeling of two types of liver injuries,and observing the heterogeneity and comparing the differences between serum samples and tissue samples.The main research results are as follows:1.HPLC-FD detection method was established based on the new probe GP-BAN,which would generate BAN and produce fluorescence after DPP-? hydrolysis.The linearity,sensitivity,recovery,precision and stability of this method have been completely verified.The lower limit of quantification is 5n M(only 2?L),which is the most sensitive method.Variance of the assay was lower than 15%.The specificity of DPP-? assay was demonstrated by using different types of single enzyme and specific inhibitors.This specificity ensures that the assay can be used for the quantitative determination of DPP-? activity in tissue microsomes,cell homogenates,cell supernatants,blood and single enzyme systems.2.In normal rats,the abundance of DPP-? was high in kidney,intestine and spleen tissues,while low in liver tissues,and the two levels were nearly 100 fold.3.Both kind of liver injury agents can significantly increase serum DPP-? level,showing a very good ability to distinguish injury.Through residual activity tests,we can basically confirm that DPP-? is not of liver origin,and is probably increased by spleen injury or proliferation.4.In order to investigate the role of different liver parts to the serum level of markers,we divided the rat liver into four parts,including lobe A(left lobe),lobe B(middle lobe),lobe C(caudate lobe)and lobe D(combined samples of left medial lobe,right lobe,papillary lobe and mastoid lobe).The differences of DPP-? activity,total tissue activity and specific activity in different tissue lobes or different parts of the liver were detected in the rat injury model made by two liver injury agents.The direct damage caused by ANIT was relatively mild,mainly due to the decrease of tissue residual activity after the damage of leaf an and the upward trend in other leaves,but none of them had statistical significance.The residual soluble DPP-? in solid tissues was removed by liver perfusion and liver washing.The results showed that the increased DPP-? in liver tissues after ANIT-induced liver injury was not soluble contamination.5.CCl₄caused more obvious direct liver injury,but no change in kidney and small intestine.In the liver,there were statistically significant changes in all liver lobes except the A lobe.At the same time,CCl₄caused a contradictory trend between the increase of specific activity of spleen DPP-? and the decrease of total activity.Although there was no statistical difference in this data,our results indicated that the immune system was inhibited by CCl₄,which needs to be further confirmed.Conclusion:In this study,we established a high sensitivity detection method for DPP-?.A method for the determination of DPP-? residual activity in serum and tissues was also established.In two recognized rat liver injury models,we found that DPP-? had a very good ability to discriminate between the two types of injury in serum samples.Combined with the detection of liver tissue residual activity,we found that the types and mechanisms of liver injury in two rat liver injury models were different.The determination of DPP-? residual activity can be used as a counter-validation marker for evaluating whether the extrahepatic tissue is injured or not,and for screening whether the injury of other extrahepatic tissues or the"second strike"or"repeated strike"of the injury occurred.Tissue residual activity is obviously not suitable for clinical use,but in basic research,examination of tissue residual activity will play a key role in promoting the clinical transformation of biomarkers.