Effects Of Baicalin On Intestinal Epithelial Cell Barrier Dysfunction Induced By Hypoxia Reoxygenation

Objective To investigate the effects of baicalin on intestinal epithelial cell barrier dysfunction induced by hypoxia reoxygenation injury. Methods Hypoxia reoxygenation injury model of rat's intestinal epithelial cell-6(IEC-6) was established, then to add fresh cell culture and baicalin, the treated cells were placed in normal incubator for 24h to do the following experiments. Optional baicalin concentration was chosen through detecting cell proliferation by using methylthiazolyl tetrazolium (MTT) assay. IEC-6 cells were randomly divided into 5 groups:normal control group(A), hypoxia reoxygenation group(B), hypoxia reoxygenation+5.0×10-3mol/L baicalin group(C), hypoxia reoxygenation+1.0×10-3mol/L baicalin group(D), and hypoxia reoxygenation+0.2×10-3mol/L baical in group(E). Supernatants were collected for determination of malondialdehyde(MDA) and superoxide dismutase(SOD).The levels of cAMP in cultured supernatans were measured by means of ELISA kit. Tight junction proteins Occludin and Zonula Occludens 1(ZO-1) expression and NF-?B protein expression were detected by using Western blot, and the expression of Occludin and ZO-1 mRNA were detected by using Reverse Transcription(RT)PCR.IEC-6 cells were seeded in transwell inserts at 2×105cell/ml. To evaluate the integrity of the mono layer, TER was performed using a Millipore electric resistance system and expressed as Ohm/well. Only inserts with a TER value>1000 Ohm/well, indicating confluence, cells were used preparing the hypoxia reoxygenation IEC-6 cells injury mode. TER was performed using a Millipore electric resistance system. Results Baicalin has inhibition effects on cell proliferation by hypoxia reoxygenation injury(normal control group absorbance value:0.442±0.015, hypoxia reoxygenation group:0.256±0.007,5.0×10-3mol/L baicalin group:0.407±0.008; P< 0.05). Compared with normal control group, the MDA level of hypoxia reoxygenation group in culture medium increased [normal control group absorbance value: (3.502±0.231)mol/L, hypoxia reoxygenation group:(5.541±0.143)mol/L], SOD level decreased [normal control group absorbance value:(20.073±0.493)×103U/L, hypoxia reoxygenation group:(13.324±0.081)×103U/L], and cAMP level increased[normal control group absorbance value:(4.575±0.122)xl0"3mol/L,hypoxia reoxygenation group:(7.28±0.143)×10-3mol/L]Occludin and ZO-1 expressions decreased, and NF-?B(P65)-. PKA expression increased (all P<0.05), TER decreased (normal control group absorbance value:1177.5±26.3Ohm/well, hypoxia reoxygenation group: 586.25±11.09Ohm/well; P<0.05). Compared with hypoxia reoxygenation group, along with baicalin concentration increasing, MDA level in baicalin groups decreased[5.0×10-3mol/L baicalin group:(4.320±0.106)mol/L,1.0×10-3mol/L baicalin group:(4.578±0.080)mol/L,0.2×10-3mol/L baicalin group:(4.928±0.118)mol/L], SOD level increased[5.0×10-3mol/L baicalin group:(18.462±0.284)×103U/L,1.0x10" 3mol/L baicalin group:(16.624±0.364)×103U/L,0.2×10-3mol/L baicalin grou: (14.023±0.140)×103U/L], cAMP level decreased[5.010-3mol/L baicalin group: (5.155±0.152)10-3mol/L,1.010-3mol/L baicalin group:(6.27±0.168)10-3mol/L, 0.210-3mol/L baicalin group:(6.948±0.199)10-3mol/L, F=211.77, P< 0.05]. protein and mRNA of occludin and ZO-1 increased, and NF-?B (P65), PKA expression decreased(all P<0.05), TER increased(5.010-3mol/L baicalin group: 1105±31.09Ohm/well,1.010-3mol/L baicalin group:790±18.26Ohm/well,0.210-3mol/L baicalin group:619.5±14.98Ohm/well, F=255.54, P<0.05). Conclusion It is suggested that baicalin alleviates the intestinal epithelial cell barrier dysfunction which induced by hypoxia reoxygenation by inhibiting NF-?B and cAMP/PKA pathways.