Alleviating penicillin-resistant Streptococcus pneumoniae‑induced lung epithelial cell injury: mechanistic insights into effects of the optimized combination of main components from Yinhuapinggan granules

Abstract

Objective: Penicillin-resistant Streptococcus pneumoniae (PRSP), for which novel treatment medicines are required, has expanded extensively due to the overuse of antibiotics. This study aimed to detect the optimal ratio of the combination of the main components based on Yinhuapinggan granules (YHPG) to generate novel treatment concepts for PRSP-induced lung injury.

Methods: Three representative main components: chlorogenic acid (C), amygdalin (A), and puerarin (P) were selected, and the optimal combination of these three components was determined by an orthogonal experiment. Investigations were conducted on the potential mechanisms underlying the protective effect of this optimized combination against PRSP-induced lung epithelial cell damage. Meanwhile, the bacteriostatic effect was further explored through the optimized combination of these natural products combined with penicillin G (PG).

Results: The optimized combination CAP (C: 16 µg/mL, A: 24 µg/mL, P: 24 µg/mL) screened by the orthogonal experimental design reduced cell damage in a model of human lung epithelial cells infected by PRSP, and the combination of CAP and PG had a synergistic effect. At the cellular level, CAP attenuated lung epithelial cell injury by modulating the TLRs/MyD88 inflammatory pathway. At the bacterial level, CAP modulated the virulence and drug resistance of PRSP, resulting in enhanced bacterial inhibition by the combination of CAP and PG.

Conclusion: Taken together, our results suggest that CAP can modulate or synergize with PG to modulate the TLRs/MyD88 pathway and attenuate PRSP-induced lung injury, and can be used as a potential drug for treating PRSP infection.

Keywords: Streptococcus pneumoniae; Drug resistance; Inflammation; Lung injury; Yinhuapinggan granules.

Fig. 1

The experimental flowchart of this study, including the corresponding main experimental methods and experimental results

Fig. 2

Effects of drugs on SP and A549 cells. (A) The MICs of PG on SP1, SP2, and SP3 were determined by AST to be 1024, 512, and 256 µg/mL, respectively. (B) The TC0 of C, A, and P on A549 cells were detected by CCK-8 assay to be 50, 520, and 320 µM, respectively. All values are presented as mean ± SD (n = 3). **p < 0.01 vs. control

Fig. 3

CAP had no significant effect on PRSP capsules but reduced PRSP autolysis activity. (A) Capsules were treated with FITC-dextran and observed using CLSM. (B) Autolysis was induced using Triton X-100 and autolysis activity was evaluated by the amount of change in OD600. All values are expressed as mean ± SD (n = 3). ##p < 0.01 vs. PBS group; **p < 0.01 vs. control

Fig. 4

100:1 was the optimal MOI; CAP attenuated PRSP-induced A549 cell damage. (A) Effects of various MOIs on A549 cells, cytotoxicity was assessed by microscopic observation (cells in 96-well plates) and LDH assay (cells in 6-well plates), respectively. (B) Effects of drugs on A549 cells after PRSP infection as measured by Hoechst 33,342/PI double staining and LDH assay. All values are expressed as mean ± SD (n = 3). ##p < 0.01 vs. control; **p < 0.01 vs. model

Fig. 5

CAP promotes the growth status of PRSP-infected A549 cells. Growth of A549 cells was monitored by RTCA. The protective effects of the drugs were also analyzed at specific time points (30 h, 36 h, 42 h, 48 h). The values in the bar graph are expressed as mean ± SD (n = 2). ##p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. model

Fig. 6

CAP attenuates PRSP-induced apoptosis in A549 cells. (A-B) The apoptosis levels were detected by flow cytometry. (C) Relative mRNA expression of Bax, Bcl-2, and Caspase-3 was analyzed by RT-qPCR. All values in the bar graphs are expressed as mean ± SD (n = 3). ##p < 0.01 vs. control; **p < 0.01 vs. model

Fig. 7

CAP reduced PRSP-induced ROS levels in A549 cells. ROS levels were detected by flow cytometry. All values in the bar graphs are expressed as mean ± SD (n = 3). ##p < 0.01 vs. control; **p < 0.01 vs. model

Fig. 8

CAP reduced the adhesion and invasion rates of PRSP on A549 cells. (A) The adhesion ability of PRSP was assessed by adhesion assay, MATH assay and RT-qPCR. (B) the invasion status of PRSP was shown by invasion assay and CLSM observation. All values are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01 vs. model or control

Fig. 9

CAP attenuates inflammatory cytokine secretion and down-regulates the expression of TLRs/MyD88-related targets in A549 cells, and also tends to down-regulate PRSP WalRK TCS-related targets. (A) TNF-α, IL-6, and IL-8 levels were analyzed by ELISA; (B) mRNA expression levels of TLR2, TLR4, and MyD88 in A549 cells & walR, walK, stkP, pcsB, and lytB in PRSP were analyzed by RT-qPCR. All values are expressed as mean ± SD (n = 3). #p < 0.05, ##p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. model

Fig. 10

WalRK TCS is involved in the maintenance of cell wall homeostasis in Streptococcus pneumoniae