Objective

2

To verify the anti-oxidative stress effect of Huangqintang based on the nuclear factor E

2

-related factor 2 （Nrf2） signaling pathway by using Caco-2 cells as a carrier and RNA interference （RNAi） technology with

in vitro

experiments.

Method

2

The Caco-2 cells in the logarithmic growth phase were transfected with siRNA to construct siRNA Caco-2 cells. After normal Caco-2 cells and siRNA Caco-2 cells were incubated with Huangqintang of different doses， RNA and protein were extracted. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the mRNA and protein expression of heme oxygenase-1 （HO-1）， NAD（P）H quinone oxidoreductase 1 （NQO1）， glutathione S-transferase （GST）， Kelch-like ECH-associated protein 1 （Keap1）， and Nrf2. Meanwhile， the activities of superoxide dismutase （SOD） and GSH-Px， as well as the expression levels of malondialdehyde （MDA） and reactive oxygen species （ROS）， were detected by the colorimetric method and the probe method.

Result

2

Compared with the results in the normal group， only the 400 mg·L

-1

Huangqintang group and the sulforaphane （SFN） group could reduce the content of ROS and MDA in Caco-2 cells （

P

<

0.01）， while the activities of SOD and GSH-Px in the cells of the Huangqintang groups and the SFN group showed an upward trend. Furthermore， there were significant differences in the 400 mg·L

-1

Huangqintang group/the SFN group and the normal group （

P

<

0.01）. Meanwhile， the protein and mRNA expression levels of HO-1， GST， Keap1， NQO1， and Nrf2 showed an upward trend in all groups （

P

<

0.05，

P

<

0.01）. After transfection， compared with the normal group， the model group showed increased content of MDA and ROS， blunted activities of GSH-Px and SOD， and reduced protein and mRNA expression of HO-1， GST， Keap1， and NQO1 （

P

<

0.05，

P

<

0.01）. After drug incubation， compared with the model group， the SFN group showed potentiated SOD activity， and the SFN group and the Huangqintang groups showed enhanced GSH-Px activity （

P

<

0.01）. Moreover， the activities of SOD and GSH-Px in the 400 and 200 mg·L

-1

Huangqintang groups and the SFN group showed an upward trend （

P

<

0.01）， and the content of MDA in the 400 mg·L

-1

Huangqintang group and the SFN group showed a downward trend. ROS decreased in all groups with drug intervention （

P

<

0.01）， and the protein and mRNA expression of HO-1， GST， Keap1， NQO1， and Nrf2 increased to varying degrees （

P

<

0.05，

P

<

0.01）.

Conclusion

2

Huangqintang can play an anti-oxidative stress role by regulating the Nrf2 pathway.