The Mitochondrial Mechanism Of Ulinastatin Improving Apoptosis Of T Lymphocytes In Rats With Severe Acute Pancreatitis

Objective:To investigate the influence of ulinastatin on apoptosis of T lymphocytes in rats with severe acute pancreatitis, confirming its direct immunoregulative activity; To elucidate the molecular mechanism of ulinastatin underlying its role of reducing apoptosis of T lymphocytes in rats with severe acute pancreatitis by analysing the effect of ulinastatin on oxidative stress level and mitochondrial apoptosis pathway in T lymphocytes.Methods:Thirty six Wistar rats were randomly divided into three groups (n=12):(1) the sham-operated control group;(2) the severe acute pancreatitis (SAP) group;(3) the ulinastatin-treated group. The severe acute pancreatitis model was established by using intrapancreatobiliary duct injection of5%sodium taurocholate. A bolus of10000U/kg Ulinastatin was intravenous injected immediately after the establishment of SAP model.24hours later, rats were sacrificed and splenic lymphocytes were collected. CD4+, CD8+T lymphocytes were labeled by using direct immunofluorescence assays, while the apoptotic cells were determined by using methods of Annexin-V/PI double-stainning. Then flow cytometry was performed to investigate the percentages and the apoptotic ratio of CD4+, CDs+T lymphocytes in rats; the intracellular production of reactive oxygen species was detected by2,7-dichlorofluoresce in diacetate (DCFH-DA) as a fluorescent probe, total superoxide dismutase levels in serum were examined by hydroxylamine colorimetric assay, and malondialdehyde levels in serum were examined by thiobarbituric acid assay; finally, the mitochondrial membrane potential levels were measured by flow cytometry with fluorescent probe rhodamine123, and mitochondria permeability transition pore opening levels were measured by coloading with calcein AM and cobalt chloride.Results:1. The percentage of CD4+T lymphocytes and the ratio of CD4+/CDg+T lymphocytes in SAP group were significantly lower than those in sham group (P<0.01), both of the apoptotic CD4+and CD8+T lymphocytes in SAP group were significantly higher than those in sham group (P<0.01), especially the CD4+T lymphocytes show more increase in apoptosis than CDs+T lymphocytes. However, ulinastatin treatment significantly attenuated the apoptosis of CD4+T lymphocytes (P<0.01), increasing the percentages of CD4+T lymphocytes and the ratios of CD4+/CD8+T lymphocytes (P<0.01);2. The intracellular production of ROS and the levels of MDA in serum in SAP group were significantly higher than those in sham group (P<0.01), but both of them significantly decreased in ulinastatin-treated group (P<0.01), while the activity of SOD in serum was significantly lower than that in sham group (P<0.01) but increased in ulinastatin-treated group (P<0.01);3. The percentage of mitochondrial permeability transition pore opening lymphocytes and mitochondrial membrane potential decreased lymphocytes in SAP group were significantly higher than those in sham group (P<0.01), while significantly reduced in ulinastatin-treated group (P<0.01).Conclusion:1. Ulinastatin can exert an direct enhancement effects on immunological function and attenuate immunosuppression in SAP rats through inhibiting the apoptosis of CD4+T lymphocytes;2. Ulinastatin can enhance the scavenging capacity of oxygen free radical, reduce the apoptotic ratio of lymphocytes, and attenuate the influence of oxidative stress on immunologic function in SAP rats through decreasing the intracellular production of ROS and the level of MDA in serum as well as increasing the activity of SOD in serum;3. Ulinastatin attenuates lymphocytes apoptosis and improves immunological function in SAP rats. It exerts the therapeutic effects through blocking the vicious cycle between ROS and mitochondrial damage, thereby inhibits the apoptosis induced by mitochondrial signaling pathways.