Modulation of P1 and EGF expression by Baicalin

Abstract

Mycoplasma pneumoniae (M. pneumoniae) is increasingly recognized as a major cause of acute respiratory tract infections. Today, macrolides are used in the primary treatment of M. pneumoniae infection. However, with the increasing prevalence of strains resistant to macrolides, as well as reports of toxicity and adverse side effects, it is necessary to develop an alternative therapeutic agent. A compound recipe - Qinbaiqingfei pellets (Qinbai) - have already been approved in China as the first effective traditional Chinese medicine to be used against M. pneumoniae. Herein, we characterize the mechanism by which Qinbai interacts with M. pneumoniae and lung epithelial cells. The fact that Baicalin is the key component of Qingbai leads us to believe its study is important to elucidating the mechanism of the action of Qinbai. In this study, we describe the complex impact of Baicalin on the adhesin protein P1 of M. pneumoniae and on the expression of epidermal growth factor (EGF) in BALB/c mice and A549 cells infected with M. pneumonia. We draw the conclusion that Baicalin not only cured M. pneumoniae infection by inhibiting P1 expression, but also enhanced the repair of lung epithelial cells by upregulating EGF. Finally, we demonstrate that Baicalin plays a role in Qinbai treatment.

Figure 1

The pathological examination of the lungs of BALB/c mice and the histology scores revealed marked differences between groups. After mice were infected with Mycoplasma pneumoniae for 3 days, they were treated with 16 μg/mL Baicalin and 100 μg/mL Qinbai for 6 days. Experiments were repeated twice.

Figure 1

The pathological examination of the lungs of BALB/c mice and the histology scores revealed marked differences between groups. After mice were infected with Mycoplasma pneumoniae for 3 days, they were treated with 16 μg/mL Baicalin and 100 μg/mL Qinbai for 6 days. Experiments were repeated twice.

Figure 2

M. pneumoniae was incubated at 37 °C with various concentrations of Baicalin and Qinbai by the serial double dilution method. MIC was 16 μg/mL for Baicalin and 100 μg/mL for Qinbai at which the metabolism of the organisms was inhibited and lack of color change was observed. Resultant mixtures are measured by real-time quantitative PCR. Baicalin and Qinbai demonstrated a reasonably good activity against M. pneumoniae. Our study suggests that Baicalin may have great potential in clinical studies.

Figure 3

P1 mRNA expression was inhibited by treatment with 16 μg/mL Baicalin and 100 μg/mL Qinbai. Real-time quantitative PCR was performed as detailed in the Materials and Methods. Experiments were repeated three times. Data are presented as mean ± SD, p < 0.01.

Figure 4

Expression of the P1 protein was assessed by immunoblot. Baicalin and Qinbai depressed P1 protein expression. Experiments were repeated twice. Data are presented as mean ± SD, p < 0.01.

Figure 5

EGF mRNA expression was increased in BALB/c mice treated with Baicalin and Qinbai. After mice were infected with M. pneumoniae for 3 days, they were treated with 16 μg/mL Baicalin and 100 μg/mL Qinbai for 6 days. Experiments were repeated three times. Data are presented as mean ± SD, p < 0.05.

Figure 6

Baicalin and Qinbai had a significant effect on EGF protein expression. Protein extracted from BALB/c mice treated with Baicalin or Qinbai was analyzed by Western blot with an antibody against EGF. Experiments were repeated twice. Data are presented as mean ± SD, p < 0.05.

Figure 7

The treatment of BALB/c mice with Baicalin or Qinbai led to an increase in EGF protein expression according to immunofluorescence results. Experiments were repeated twice.

Figure 8

We performed reverse transcription-PCR to analyze the levels of EGF mRNA. After A549 cells were infected with M. pneumoniae for 4 h, cells were treated with 16 μg/mL of Baicalin and 100 μg/mL of Qinbai for 3 days at 37 °C. EGF expression in A549 cells was increased by Baicalin and Qinbai. Data are presented as mean ± SD, p < 0.05.

Figure 9

The treatment of cells with Baicalin or Qinbai led to an increase in EGF protein expression according to immunoblotting. Data are presented as mean ± SD, p < 0.05.

Figure 10

Immunofluorescence was used to analyze EGF protein expression. Experiments were repeated twice.