Protective Effect of Distillate and Redistillate of Cow’s Urine in Human Polymorphonuclear Leukocytes Challenged With Established Genotoxic Chemicals

Objective From the ancient period cow’urine has been used as a medicine. In Veda, cow’urine was compared to the nectar. In Susrut, several medicinal properties of cow’ urine have been mentioned and are known to cause weight loss, reversal of certain cardiac and kidney problems, indigestion, stomach ache, edema, etc. However, the literature and scripture did not mention the antigenotoxic properties of cow’urine. Methods In the present investigation, the antigenotoxic/ antioxidant properties of cow’ urine distillate and redistillate were studied in vitro. The antioxidant status and volatile fatty acid levels were determined. Actinomycin-D (0.1ol/L) and hydrogen peroxide (150 mol/L) were used for inducing DNA strand break with 0.1% DMSO as negative control. Dose for the antigenotoxic effect of cow’ urine was chosen from the dose response study carried out earlier. Results Both actinomycin-D and H2O2 caused statistically significant DNA unwinding of 80% & 75% respectively (P<0.001) as revealed by fluorimetric analysis of DNA unwinding (FADU), and the damage could be protected with the redistilled cow urine distillate (1, 50 & 100 ) in simultaneous treatment with genotoxic chemicals. Conclusion The redistillate of cowurine was found to possess total antioxidant status of around 2.6 mmol, contributed mainly by volatile fatty acids (1500 mg/L) as revealed by the GC-MS studies. These fatty acids and other antioxidants might cause the observed protective effects.

Expression of platelet endothelial cell adhesion molecule-1 between pancreatic microcirculation and peripheral circulation in rats with acute edematous pancreatitis

OBJECTIVE: To study the ehanges of platelet endothelial cell adhesion molecule-1 (PECAM-1) expression on polymorphonuclear leukocytes (PMNs) in peripheral circulation anti pancreatic microcirculation in rats with acute edematous pancreatitis (AEP). METHODS: The model of AEP was established with 50 Wistar rats, and the changes of PECAM-1 expression on PMNs from the splenic vein and inferior vena cava were determined by flow cytometry. RESULTS: PECAM-I expression on PMNs showed no significant difference between pancreatic microcirculation and peripheral circulation at AEP2h and AEP4h time points. From the AEP4h to the AEP8h time point, PECAM-1 expression in peripheral circulation was up-regulated, but PECAM-1 expression in pancreatic microcirculation was down-regulated. PECAM-1 expression had a significant difference between pancreatic microcirculation and peripheral circulation at the AEP8h time point (P<0.05). CONCLUSION: PECAM-1 expression on PMNs is in a converse way between pancreatic microcirculation and peripheral circulation in AEP.

High-mobility group box1 as an amplifier of immune response and target for treatment in Aspergillus fumigatus keratitis

AIM:To determine the roles of high-mobility group box1(HMGB1)in pro-inflammation,host immune response and its potential target for treatment in Aspergillus fumigatus(A.fumigatus)keratitis.METHODS:Expression of HMGB1 was tested in C57 BL/6 normal and infected corneas.Dual immunostaining tested coexpression of HMGB1 with TLR4 or LOX-1.C57 BL/6 mice were pretreated with Box A or PBS and then infected.Clinical scores,polymerase chain reaction,ELISA,and MPO assay were used to assess the disease response.Flow cytometry were used to test the effect of Box A on reactive oxygen species(ROS)expression after A.fumigatus stimulation in polymorphonuclear neutrophilic leukocytes(PMN).C57 BL/6 peritoneal macrophages were pretreated with Box B before A.fumigatus stimulation,and MIP-2,IL-1β,TNF-α,HMGB1 and LOX-1 were measured.Macrophages were pretreated with Box B or Box B combined with Poly(I)(an inhibitor of LOX-1)before stimulating with A.fumigatus,and MIP-2,IL-1β,TNF-α,LOX-1,p38-MAPK,p-p38-MAPK were measured.RESULTS:HMGB1 levels were elevated in C57 BL/6 mice after infection.HMGB1 co-expressed with TLR4,and LOX-1 in infiltrated cells.Box A vs PBS treated C57 BL/6 mice had lower clinical scores and down-regulated corneal HMGB1,MIP-2,IL-1βexpression and neutrophil influx.Box B treatment amplified expression of MIP-2,IL-1β,TNF-α,HMGB1 and LOX-1 that induced by A.fumigatus in macrophage.Compared to the treatment of Box B only,the protein expression of IL-1β,TNF-αshowed inhibition of Box B combined with Poly(I),which also reduced the A.fumigatusevoked protein level of LOX-1 and phosphorylation level of p38-MAPK.The production of A.fumigatus-stimulated ROS was significantly declined after Box A pretreatment in PMN.CONCLUSION:Blocking HMGB1 reduces the disease response in C57 BL/6 mice.HMGB1 can amplify the host immune response through p38-MAPK,and is a target for treatment of A.fumigatus keratitis.

The effects of amniotic membrane on polymorphonuclear cells

Objective To investigate the effects of fresh and preserved amniotic membrane on polymorphonuclear neutrophils (PMNs) so as to understand the anti-inflammatory mechanism of amniotic membrane transplantation.Methods Conditioned medium was collected 48 hours after fresh or preserved amnions were cultured in DMEM and 5% CO 2 at 37℃. Then, polymorphonuclear cells were cultured in conditioned culture or DMEM. Fluorescent microscopy with 4’,6-diamidino-2-phenylindole (DAPI) staining and cytometry were performed 6, 9, 12, and 15 hours later. Results Apoptotic neutrophils were found in each group at different time points. The percentage of apoptotic cells at 6, 9, 12, and 15 hours after culture in the fresh and preserved amnion groups and the control group was 17.3%, 24.4%, 29.8%, 37.1%, and 16.2%, 20.1%, 23.7%, 27.7%, and 10.2%, 13.7%, 21.1%, 26.4%, respectively (t test, P 1<0.01, P 2<0.01 and P 3<0.01).Conclusion Amniotic membrane can accelerate apoptosis of polymorphonuclear neutrophils, reduce inflammation, and prevent ocular surface collagen from resolution, indicating that fresh amnion might have a stronger effect than preserved amnion.