Study On The Antioxidative Stress Of Huangqin Tang On Ulcerative Colitis Through The Nrf2 Pathway

Huangqin Tang(HQT) is a famous prescription in Shanghan Lun, which is consist of Huangqin, Shaoyao, Gancao and Jujube and commonly used to heat dysentery and relieve pain.In present, it is widely used in the treatment of bacillary dysentery, amebic dysentery, acute gastroenteritis, ulcerative colitis and other gastrointestinal diseases in clinical practice.Modern pharmacological studies have shown that HQT has significant anti-inflammatory, antipyretic, anticancer and protecting liver, but the mechanism is unclear.In this paper, as a guidance for the Chinese medicine theory, the use of modern technologies, from the whole animal, cellular and molecular different levels, using the rat model of ulcerative colitis and cell model RNAi Caco-2, study the HQT anti-oxidative stress mechanism of preliminary study and provide scientific basis for clinical application and further development skullcap soup.Objective:1、To build the model of ulcerative colitis rats, and observe the therapeutic effect of decoction skullcap to clear antioxidant HQT stress effect.2、To establish and determine the model of Caco-2 interfered by siRNA Nrf2.3、To observe the effect of HQT on the model of Caco-2 interfered by siRNA Nrf2 and study the antioxidative stress through the Nrf2 pathway.Methods:1、The treatment of HQT on oxidative stress in rats with ulcerative colitisThe model of UC rats with cell immunoreactivity were made using compound method which mixed the Trinitrobenzene sulfonic acid and ethanol. Rats were randomly divided into control group, model group, SASP group, high dose, middle dose and low dose of HQT. Combining our previous study with the food intake, weight and spirit, we determine the successful model. The SASP, HQT or saline were adminnistraed for 5 days after being made successfully. The blood and colon were also collected. Production of GSH-px, CAT, SOD and MDA were measured by biochemical assay, the expression level of LPO were determined by ELISA. ICH method was undertaken to determine the expression of Nrf2 protein in colon tissue. Meanwhile, western blot method was undertaken to test the expression of HO-1, NQO1 in colon tissue.2、The effect of HQT on oxidative stress in Caco-2 cellsThe cells in exponential growth period were cultured in 6 well plate and interfered with HQT or SFN for 36h.Clear the supernate and extract the RNA with Trizol. The expressions of HO-1, NQO1 and GST mRNA were tested by RT-PCR. Meanwhile, extract the protein with Protein Lysis Buffer. Then the production of SOD, MDA, ROS were detected by related methods and the expressions of HO-1, NQO1, GST protein were tested by western blot.3、The effect of HQT on oxidative stress in siRNA Nrf2 Caco-2 cells3.1 The construction and identification targeting siRNA Nrf2 gene in Caco-2 cellsOn the basis of principle on Tuschl, according to the sequence of Nrf2 in Genebank,3 pairs of siRNA Nrf2,1 pair of negative control and GAPDH positive control were synthesis by Genephama. Before the transfection, cells were cultured in 6 well plate with a number of 2×105.The Lipofectamine2000 and the siRNA were integrated in a well. Then the mixture was transfected into Caco-2 and was cultured for 36h. The expression of Nrf2 mRNA was measured by RT-PCR and the expression of Nrf2 protein was measured by Western blot.3.2 The effect of HQT on oxidative stress in siRNA Nrf2 Caco-2 cellsThe cells in exponential growth period were cultured in 6 well plate. According to the tranfection for 6h, the cells were interfered with HQT or SFN for 36 h. Clear the supernate and extract the RNA with Trizol. The expressions of HO-1, NQO1 and GST mRNA were detected by RT-PCR. Meanwhile, extract the protein with Protein Lysis Buffer. Then the production of SOD, MDA, ROS were detected by related methods and the expression of HO-1, NQO1, GST protein were measured by western blot.Results:1、The treatment of ulcerative colitis and antioxidative stress of Huangqin Tang The food intake and body weight of model group rats were lower than that of control group. And the rats of model group exist in hematochezia, diarrhea, lazy behavior, dark hair and ulceration colonic tissue. After treatment of SASP and HQT, the spirit of each treated group have a different development. Compared with the model group, the food intake and weight have a significant increase. The production of SOD, CAT, GSH-px, LPO are higher than those of the model group in serum and the level of MDA in serum in model group decrease. Meanwhile, the expression of Nrf2, HO-1, NQO1 in colon were higher than those of model group.The above mentioned indexes in model group have a significant distinction than those of normal group.2、The effect of HQT on oxidative stress in Caco-2 cellsCompared with the normal group, the groups of SFN and HQT 400μg/ml can decrease the production of ROS, MDA, which have a significant difference. The groups of SFN and each HQT group are all increase the content of SOD, which shows that the SFN and 400μg/ml groups have a significance. Meanwhile, the production of every treat group are higher than those of model group. In addition, after the treatment, the expression of GST, HO-1, NQO1, Nrf2 mRNA and their protein are all increasing. Those results show that HQT have a certain antioxidative stress on the treatment of ulcerative colitis by the Nrf2 pathway.3、The effect of HQT on oxidative stress in siRNA Nrf2 Caco-2 cells3.1 The construction and identification targeting siRNA Nrf2 gene in Caco-2 cellsAfter transfected with Nrf2 siRNA, compared with the normal group, three pairs of siRNA have an obvious inhibition, which the results of RT-PCR show that their inhibition ratio were 60.55%,70.83%,45.33%. Because of the highest inhibition rate of Nrf2-821, the sequence of it is deemed as targeting sequence. What’s more, the results of western blot have a similar tendency. These results show that we successfully creat the construction of siRNA Caco-2 cells targeting the Nrf2 and provide experimental basis for the targeting treatment of HQT.3.2 The effect of HQT on oxidative stress in siRNA Nrf2 Caco-2 cellsAfter tranfected with Nrf2 siRNA, compared with the normal group, SOD, MDA, ROS, GST, HO-1, NQO1,Keap1,Nrf2 of the model group have a different variation. After the treatment, the production of ROS,MDA in each treat group have a certain decrease, the production of SOD increase. Meanwhile, the reaults of western blot show that the expressions of GST, HO-1, NQOl,Ceap1,Nrf2 increase. The results of RT-PCR show that the expressions of GST, HO-1, NQO1, Keapl,Nrf2 mRNA increase. All of those results indicate that the Nrf2 pathway play an important role in the antioxidative stress and HQT have a certain antioxidative stress on the production of phase II detoxification enzymes and antioxidants by the Nrf2 pathway.Conclusion:1、HQT plays an vital role in the antioxidative stress through increasing the of phase II detoxification enzymes and antioxidants, such as the SOD, CAT, GSH-px, GST, HO-1, NQO1 and so on, and decreasing the production of MDA.2、HQT can active the Nrf2 pathway to up regulate the expressions of phase Ⅱ detoxification enzymes and antioxidants.3、Successfully construct the model of siRNA targeting Nrf2 Caco-2 cells and HQT can active the Nrf2 pathway to upregulate the expression of phase II detoxification enzymes and antioxidants on the siRNA targeting Nrf2 Caco-2 cells.