Experimental Research On The Change Features Of Traumatic Condition On Pancreatic Trauma Model In Wistar Rat And The Inhibition On PLA₂ And Protection Effects For The Model Of Variabilin

Study background and purpose The incidence of pancreatic trauma (PT) is 0.4/100,000, and it only occupies 1%-3% in abdominal trauma. However, the mortality of PT is highly to 20%,and the incidence of severe complications,such as pancreatic fistula, intestinal fistula, infection of abdominal cavity and bleeding and so on, is 40%. Therefore PT plays an important part in abdominal trauma .At present our study focus on surgical remedy after trauma, prevention and cure to trauma complications, and the study on the mechanism of traumatic conditions variation is severely deficient.The purpose of the study on variation of traumatic conditions in single organ help us to eliminate the influence of other organs'trauma, better understand its pathogenesis after trauma, master the evolutional features of traumatic conditions, and improve its therapeutic efficacy. The traditional animal models of PT mostly have such problems, for instance, complex wound agents, the out of controlled trauma range, mal-reproducibility and complicated performance. Consequently, to found a pure animal model of PT and study its pathologic and physiopathologic development becomes an emergent topic. Meanwhile, what evolutional features will be after PT? And if these features are conformity with those of acute pancreas(AP)?Phospholipase A₂(PLA₂) is a generic name of a group of enzyme which can selectively break in Sn-2 point of the phospholipids'glycerin part. The activated PLA₂ can affect the glycerophospholipide of cell membrane and mitochondria membrane, and decomposing it into lysolecithin at AP. So those construction of lipoprotein are destroyed and cells die.Then is PLA₂ activated at PT likewise at AT? How does the expressing levels of PLA₂ gene and protein, and where does the PLA₂ protein distribute in pancreas at PT?Now there are no specific enzyme inhibiters against PLA₂ for clinical using. Variabilin, which first refined from sponge in 1973, was verified to be a specific active inhibitor against PLA₂.The problem of source was successfully solved after people obtained Variabilin by chemosynthesis method. However, whether Variabilin can inhibit the activity of PLA₂ in vivo and take the protective effect?Methods For answering these formal questions, we designed our experiments as follows: first is founding a model of PT in Wister rats by BIM-â…¢biological striking machine, studying its pathologic change and routine enzyme variation at variably powers with different pressures, and get stable trauma pressure of model founded. Second is studying the traumatic condition changes in a choosey pressure at different post-PT phases. Moreover, we detected model PLA₂ activity, express of PLA₂ subtype mRNA by fluorescent quantitative RT-PCR technique, protein express and site by Western blot and IHC at different post-PT phases. At last, we studied the PLA₂ inhibited effects and model protection of Variabilin in vivo.Results and Conclusion1. We found that the survival rate is of 85% at the 24 hours phase after the impact with 400 kPa effect, and it is obviously higher than that of impact with 600 kPa, which is only of 40%.So,the upper limit of model founding pressure is 400 kPa which can be more economically applied to the observation of features of traumatic conditions in subacute stage after trauma.2. The general pathologic changes: pancreas bleeding and blood ascites formation at the strike affecting moment, and the pressure of strike is of direct ratio with blood ascites quantity and pancreas weight. Light microscope: vacuolization, edema, necrosis index of pancreas cells were serious in the groups which pressures were more than 400 kPa, and these were similar with those of AP. But few macrophage infiltration and obviously bleeding were different with those of AP. Electron microscope: endocytoplasmic reticulums expanding and breaking, ribosomes defluxion, lysosomes increasing, vascular endothelial cells swelling and blood vessels congestion appeared in all groups, and the mitochondria swelling is serious in the groups which pressures were more than 400 kPa.3. The AMS, LPS activities in blood and ascitic of experiment groups were obviously high than those of control groups at 24h phase after PT.AMS activities were no obviously difference in any of experiment groups, and LPS activities of hyper-400 kPa groups were higher than those of hypo-400 kPa, and all were different with those of control group. So we can say, AMS is a kind of indication of PT diagnosis other than prediction of PT severity degree. Meanwhile if LPS is obviously high, which can predict the existence of severe pancreatic trauma.4. After PT with 400kPa pressure, the volume of blood ascites was at the top at 6h, and decreased with time gone. Pancreas wet weight was to peak at 24h, after this was falling off. Light microscope: hemorrhage, vacuolization, edema and necrosis were the most notable changes, but inflammatory cells infiltrating was few. These above pathology indexes were to peak at 6-24h, after this fell off and recovered at 7d. Electron microscope: rough endoplasmic reticulum, ribosome, mitochondria, lysosome and capillary endothelium cell, catheter intercells changed early and began to recover after 72h.5. Blood WBC and W-LCR increased after 6h, and were to peak at 24h, then decreased. RBC, HGB and Ca in blood were decreasing since 72h after PT, but increased slightly at 7d. Blood AMS increased at 6h, and hit the peak at 24h, then decreased after this. Blood LPS also increased at 6h, but its peak time was 72h, and was at high levels at 7d.While ascites AMS and LPS were both at high level after 7d.Clinical significance: Blood AMS can be the early diagnosis criteria. Blood LPS can be the specific diagnosis criteria. But AMS and LPS can't be the criteria of prognostic judgment, only be the indexes of auxiliary diagnosis.6. Apoptosis appeared at 6h and it was the peak time by TUNEL method and FCM technology detection. The condition of PI decreasing at that time illustrated that cells were dying at the beginning after PT. After 24h, AI was decreasing and PI was increasing, and was to peak at 7d.Those demonstrated that the cell repair mechanism was activated at 24h, and it lasted to the later period with destroy mechanism after PT.7. The activity of PLA₂ in blood and pancreas tissue increased at 6h after PT, and its peak time was 24h, then deceased but still to remain higher level than that of control group. This change is more obvious in pancreas than blood. For the activity of PLA₂ in blood also increased, we can detect it from 6h to 7d after PT.8. The level of mRNA and protein expressed were same: the most increased gene was sPLA₂ after PT, then cPLA₂, last one was iPLA₂.All subtypes of PLA₂ were increasing at early after PT. The peak time of cPLA₂ and sPLA₂ was 24h, but iPLA₂ was 72h.The reason maybe is cPLA₂ and sPLA₂ are mostly produced in pancreas, but iPLA₂ is out it.9.The organization fixing of apoenzyme subtypes by IHC: The most increasing expressing were cPLA₂ and sPLA₂ in pancreas, they were found all over in the pancreatic acinar cells in 6h after PT, and spread out from the cells in 24h,then decreased after 72h.But the changes of iPLA₂ organization fixing wasn't obviously. Associated with the above experimental results, we can say iPLA₂ isn't the chief causative agent after PT.10. Afer the sponge extractive, Variabilin was applied to the PT model, we found that the activities of PLA₂ in serum and pancreas tissue were inhibited by Variabilin at 24h after trauma, the pancreas tissue swell was lessened under CT, and the morphous and organelle of pancreatic cell were protected by Variabilin under the light and electron microscope. Variabilin can inhibit the activities of PLA₂ in serum and pancreas tissue in vivo and has protective function against acute pancreatic trauma in rat by intra-abdominal administration.In a word, the main results of our study are as follows:1. The pancreatic trauma model in Wistar rat was founded. It is efficient, fine repeatable, economic and ideal for pathologic and therapeutic study further.2. The period from 6h to 24h after PT is the traumatic condition aggressive stage. the cell repair mechanism was activated at 24h,and it lasted to the later period with destroy mechanism after PT.3. The activity of PLA₂ in blood and pancreas tissue increased after PT, mostly for the cPLA₂ and sPLA₂ produced a marked effect.4. Variabilin can inhibit the activities of PLA₂ in serum and pancreas tissue in vivo and has protective function against acute pancreatic trauma in rat by intra-abdominal administration.