Qingfei Tongluo formula mitigates Mycoplasma pneumoniae infection via PERK signaling pathway

doi:10.21203/rs.3.rs-1410042/v1

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Qingfei Tongluo formula mitigates Mycoplasma pneumoniae infection via PERK signaling pathway

https://doi.org/10.21203/rs.3.rs-1410042/v1

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Introduction: Mycoplasma pneumoniae pneumonia (MPP) is usually found in school-aged children and seems to relapse easily because of the resistance to antibiotics. Qingfei Tongluo formula (QTF) is clinical used Traditional Chinese Medicine to treat MPP. Our previous study demonstrated that QTF exhibited ameliorative effects on experimental MPP mice model. In this study, we tried to further explore the function and underlying mechanism of QTF in MPP.

Methods: MP was applied to infect A549 cells and BALB/c mice to mimic MPP in vitro and in vivo. Cytokine release and reactive oxygen species (ROS) production were analyzed using Enzyme-linked immunosorbent assay (ELISA) assay and flow cytometry. Western blot analysis was used to detect the protein involved in endoplasmic reticulum (ER) stress.

Results: We found that MP infection enhanced cytokine release and ER stress in vitro and in vivo, and these effects could be alleviated by QTF. Moreover, protein kinase RNA-like endoplasmic reticulum kinase (PERK) knockdown alleviated MP infection-induced cytokine release, ROS production, and ER stress in A549 cells. While PERK overexpression exhibited the opposite effects.

Conclusions: QTF alleviated MP infection-induced cytokine release, ROS production, and ER stress via inhibiting PERK signaling pathway.

Traditional Chinese Medicine

ER stress

pneumonia

Respiratory tract infections have been and remain a major public health concern. It can range in severity from mild to life-threatening, especially dangerous in children(1). Mycoplasma pneumoniae (MP), a common bacterial pathogen, is considered as an important cause of respiratory tract infections(2). MP is also associated with community-based pneumonia, particularly among school-aged children and young adults. Due to the resistance to antibiotics, MP-induced pneumonia (MPP) becomes easy to relapse. Therefore, new effective drugs are needed. Traditional Chinese Medicine attracts researchers’ attention due to their increased safety and effectiveness.

Qingfei Tongluo formula (QTF), a Traditional Chinese Medicine, is developed by pediatricians at Longhua Hospital (Shanghai, China). It has been widely used to treat MPP with azithromycin (AZM) in clinic, showing a good curative effect. QTF is composed of the following Chinese medicines—cortex mori from Morus alba L. (9 g), cortex lycii from Lycium chinense Mill. (9 g), peach kernel from Prunus persica L. (9 g), aidicha from Ardisia japonica (Thumb.) Blume (9 g), lumbricus from Pheretimaas pergilum (E Perrier) (9 g), almond from Amygdalus communis Vas (9 g), seed from Perilla frutescens (L.) Britt. (9 g), semen lepidii from Heleocharis dulcis (Burm. f.) Trin. (9 g), and liquorice from Glycyrrhiza uralensis Fisch. (3 g). Our previous study reported that QTF exhibited ameliorative effects on mycoplasma pneumonia mice model(3). Furthermore, we demonstrated that QTF treatment showed better ameliorative effects than azithromycin in the patients with MPP(4). However, the underlying mechanism remains largely unknown.

The endoplasmic reticulum (ER), an important organelle in eukaryotic cells, is responsible for the synthesis and folding of proteins and storage of free calcium(5). Some physiological stress including Ca²⁺ levels, disturbances in redox, or other environmental factors induce the accumulation of misfolded proteins leading to ER stress. The ER stress, triggering the unfolded protein response (UPR), mainly involves three signaling pathways: inositol-requiring enzyme-1α (IRE1α), activating transcription factor 6 (ATF6), and protein kinase RNA-like endoplasmic reticulum kinase (PERK)(6). In addition, UPR is associated with several major cellular activities including pro-inflammatory response, autophagy, apoptosis, innate immunity, and the mitogen-activated protein (MAP) kinase pathways(7).

In the current study, QTF was applied to treat MP infected A549 cells and BALB/c mice. The results indicated that QTF played a protective role in MP infection in vitro and in vivo.

MP culture

MP, ATCC15531 strain (American Type Culture Collection, Rockville, MD, USA) was cultured in modified Hayflick medium (GZBIOTEST Co., Ltd, Guangdong, China) supplemented with horse serum, PPLO broth, 25% yeast extract, 0.5% glucose, 0.025% thallium acetate, penicillin G (1,000 U/ml), and 0.002% phenol red, pH 7.6, at 37 °C, 5% CO₂ for 7 days.

Cell culture and infection

A549 and 293T cells were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco,) at 37°C 5% CO₂.

For MP infection, culture medium was replaced with MP infection medium containing 10% MP (approximately 1 × 10⁷ CFU/10⁶ cells) unless otherwise specified, and cultured for 12, 24, and 48 h. Culture medium containing only mycoplasma broth without bacteria was used to culture control cells.

Enzyme-linked immunosorbent assay (ELISA)

The contents of IL-8 and TNF-α were determined with ELISA kit (Jiancheng, Nanjing, China) according to the manufacturer’s protocol.

Real-time PCR

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. One microgram of total RNA was used to generate cDNAs using the Revert AidTM First Strand cDNA Synthesis Kit (#K1622; Thermo Fisher Scientific Inc, Grand Island, NY, USA) with random primer. Then, real-time PCR was performed to quantify mRNA levels with SYBR Green PCR Master Mix (#K0223; Thermo) on an ABI 7300 thermocycler (Applied Biosystems, Foster City, CA, USA). The relative gene expression levels were quantified using 2^-^Δ^Ct method and β-actin was used as an internal control. The Primers were as follows:

PERK Primer F 5' ATCGCAGAGGCAGTGGAG 3'

Primer R 5' CATTGGGGTCAGAACCGT 3'

IRE1α Primer F 5' GAGGACAGGCTCAATCAA 3'

Primer R 5' GGTCAGGAGGTCAATAACA 3'

ATF6 Primer F 5' TTCCGTGACTAAACCTGT 3'

Primer R 5' TTAATCTCGCCTCTAACC 3'

β-actin Primer F 5' TGGCATTGCCGACAGG 3'

Primer R 5' GCATTTGCGGTGGACG 3'

QTF extract

All TCM were purchased from Shanghai Huayu Chinese Herbs Co. Ltd. including bark of Morus alba L. (XD14122403, Henan Province), bark of Lycium chinense Mill. (LY1501125, Shanxi Province), peach kernel from Prunus persica L. (XD14082202, Hebei Province), whole plant of Ardisia japonica (Thumb.) Blume (LY1410211, Henan Province), Pheretimaas pergilum (E Perrier) (LY1412060, Shanghai City), almond from Amygdalus communis Vas (XD14071106, Hebei Province), seed of Perilla frutescens (L.) Britt. (YT14121701, Gansu Province), Heleocharis dulcis (Burm. f.) Trin. (DH14092405, Shandong Province), and rhizome of Glycyrrhiza uralensis Fisch. (HY14112503, Gansu Province). The QTF components (250 g) were pulverized with a motor-driven grinder to prepare the extract. Then the extract was refluxed twice with distilled water (2 L) for 1 h each time, followed by filtration, centrifugation, and evaporation to dryness under reduced pressure in a rotary evaporator. The yield was 13.6 %.

Evaluation of reactive oxygen species (ROS)

Cells were harvested and probed with 10 μM of 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime, Shanghai, China) at 37°C for 20 min, followed by analysis with a flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Western blot

Total cells and tissues were prepared with RAPI buffer containing proteinase inhibitor (Pierce, Rockford, IL, USA). NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) was used to separate the cytosolic fraction and nuclear extracts. . Supernatant samples were separated by 10% or 15% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Billerica, WI, USA). After blocking with 5% non-fat milk, the membranes were probed with primary antibodies (anti-PERK, Abcam, Ab229912; anti-eIF2, Abcam, Ab169528; anti p-eIF2, Abcam, Ab32157; anti-ATF4, Proteintech,10835-1-AP; anti-CHOP, Proteintech,15204-1-AP; anti-β-actin, Proteintech, 66009-1-lg; anti-NF-κB, Abcam, Ab16502; anti-H3, Proteintech,17168-1-AP) at 4°C overnight. The second day, after washing away the unbound antibody, the membranes were washed and incubated with HRP-conjugated rabbit secondary antibody (Beyotime) at room temperature for 1 h. The signals from the immunoreactive proteins were detected using the ECL reagent (Millipore).

Lentivirus preparation

Short hairpin RNA (shRNA) oligos targeting PERK were annealed and cloned into AgeI- and EcoRI-digested pLKO.1 (Addgene, Cambridge, MA, USA). The full-length human PERK was cloned into pLVX-puro (Clontech, Palo Alto, CA, USA). The lentivirus was produced in 293T cells along with packaging plasmids, psPAX2 and pMD2.G.

MP infection mouse model

Four-week-old BALB/c mice (15 ± 1 g) were obtained from Shanghai Sippr Bk Laboratory Animals Co. Ltd. (Shanghai, China). All mice were randomly divided into five groups: control, model, QTF low dose, QTF middle dose, and QTF high dose (n=6). Control group mice were treated with 100 μL normal saline by nasal drip. All the other groups were treated with nasal drops containing 100 μL MPFH (1 × 107 ccu/mL) for 2 days. Afterwards, 0.25 mL normal saline per 20 g body weight were orally given to the mice in the control and model groups by gavage once a day. QTF groups were orally given 0.425, 0.85, and 1.7 g/kg QTF for 3 consecutive days, respectively. Ethical approval for the study was provided by the independent ethics committee of Shanghai University of Traditional Chinese Medicine. The inferior lobe of the right lung was harvested on the fifth day and fixed with 4% paraformaldehyde, embedded with paraffin, and cut into slices for hematoxylin and eosin (H&E) staining. Morphometric analysis was performed using an optical microscope (Leica DMLB, Germany). Alveolar lavage fluid was harvested to detect the release of IL-8 and TNF-α.

Statistical analysis.

All the experiments were conducted at least three times. Data were expressed as means ± standard deviations. Statistical analysis was performed by using ANOVA test. A P value of <0.05 indicated a significant difference.

MP infection induces cytokine expression and ER stress

Firstly, MP was applied to A549 cells and cultured for 12, 24, and 48 h. As shown in Figure 1A, MP infection increased the production of TNF-α and IL-8 in a time-dependent manner. ATF6, PERK, and IRE1α are the 3 major signaling pathways involved in ER stress, thus we detected the levels of ATF6, PERK, and IRE1α using real time PCR. The results illustrated that the mRNA levels of PERK and ATF6 were significantly upregulated upon MP infection. However, there was no significant change in IRE1α level (Figure 1B), indicating IRE1α signaling pathway may not affect by MP infection.

QTF alleviates MP infection-induced cytokine release, ROS production, and ER stress in A549 cells

To explore the function of QTF, different concentrations (0, 0.1, 0.2, and 0.4 mg/mL) of QTF were applied to MP infected A549 cells. ELISA assay showed that the increase of IL-8 and TNF-α induced by MP was inhibited after treatment with MP in a dose-dependent manner (Figure 2A). Nuclear factor-κB (NF-κB), a common anti-inflammatory target, has been related to the production of proinflammatory cytokines such as TNF-α and IL-8(8). As shown in Figure 2B, the nuclear NF-κB level was promoted after MP infection but depressed upon treatment with QTF. Opposite effects were observed of the cytoplasmic NF-κB level, indicating MP induced the NF-κB activation could be inhibited by QTF. Furthermore, ROS have been reported to regulate the activity of NF-κB, so we detected ROS level upon treatment with MP and QTF. As expected, MP infection increased ROS level in A549 cells, however, cells treated with MP and QTF exhibited lower ROS level than the cells treated with MP only (Figure 2C). In addition, the increase levels of PERK and ATF6 induced by MP were alleviated after QTF treatment (Figure 2D), suggesting that PERK and ATF6 signaling pathways were involved in function of QTF in MP treated cells. As shown in Figure 2E, MP infection upregulated the protein levels of PERK, p-eIF2α, ATF4, and CHOP. QTF could decreased these protein levels in a dose-dependent manner. Taken together, QTF mitigated the effects induced by MP infection in A549 cells.

Silencing of PERK alleviates MP infection-induced cytokine release, ROS production, and ER stress in A549 cells

To confirm whether PERK was involved in MP infection, knockdown lentiviruses targeting PERK were applied to A549 cells upon MP infection. As shown in Figure 3A, three shPERK lentiviruses significantly decreased the protein level of PERK in A549 cells. Furthermore, we found that PERK silencing could alleviate MP infection-induced the release of TNF-α and IL-8 (Figure 3B), NF-κB activation (Figure 3C), and ROS production (Figure 3D). In addition, the increase of PERK, p-eIF2α, ATF4, and CHOP levels induced by MP infection was inhibited by PERK silencing (Figure 3E). These results further confirmed that PERK signaling pathway played a role in MP infection.

QTF alleviates PERK overexpression-induced cytokine release, ROS production, and ER stress in A549 cells

Firstly, PERK overexpressing lentivirus was used to enhance PERK expression. As expected, protein level of PERK was markedly upregulated by PERK overexpressing lentivirus (Figure 4A). Based on the results in Figure 3C, shPERK depressed MP infection-induced activation of NF-κB. Then, PDTC, an NF-κB inhibitor, was applied to A549 cell. As shown in Figure 4B, PERK overexpression promoted the release of TNF-α and IL-8, however, co-treatment with PERK overexpressing lentivirus and PDTC showed lower level of TNF-α and IL-8 than that in the cells treated with PERK overexpressing lentivirus alone. Moreover, PERK overexpression could increase the release of TNF-α and IL-8 (Figure 4C), NF-κB activation (Figure 4D), ROS production (Figure 4E), and the protein levels of PERK, p-eIF2α, ATF4, and CHOP (Figure 4F). But these effects were mitigated by QTF in A549 cells. In summary, these results indicated that QTF played a protective role in A549 cell via PERK signaling pathway.

QTF mitigates MP infection-induced cytokine release and ER stress in vivo

To further explore the function of QTF, MP infection mouse model was established and treated with low, middle, and high dose of QTF. HE staining showed that pretty normal lung tissues were observed in control group. In the model group, there were many pulmonary interstitial infiltrations of lymphocytes and plasmacytes as well as bronchus and vasodilation congestion. QTF relieved the injury induced by MP infection in a dose dependent manner (Figure 5A). Furthermore, we found that QTF alleviated MP infection-induced the release of TNF-α and IL-8 (Figure 5B), NF-κB activation (Figure 5C), and the protein levels of PERK, p-eIF2α, ATF4, and CHOP (Figure 5D) in vivo, indicating that QTF played a protective role in MP infection mouse model.

MP infection is the cause of several diseases including pneumonia. As reported, the incidence of MPP in children is increasing(9). Researchers prefer to focus on exploring the mechanism under antibiotic treatment of MPP. The effects of Traditional Chinese Medicine were usually ignored. QTF was firstly developed by the Pediatric Department at Shanghai Longhua Hospital and exhibited an effective role in the treatment of children with MPP(10). In our previous study, we found that QTF showed ameliorative effects on MPP mice model(3). In the current study, we demonstrated that QTF alleviated MP infection-induced cytokine release, ROS production, and ER stress in vitro and in vivo.

When infection happens, the first response of our body should be the inflammation caused by innate immune. TNF-α and IL-8 are the two important cytokines of inflammation process. TNF-α, which is produced by monocytes and macrophages, plays a key role in MP infection induced lung injury(11). Higher serum level of TNF-α is observed in children with MPP(12). Recently, high expressions of both TNF-α and community-acquired respiratory distress syndrome toxin are considered to be a good predictor for MPP(13). MP has been found to induce the production of IL-8 in bronchial epithelial cells via NF-κB or ERK signaling pathway(14). In this study, we found that MP infection enhanced the release of TNF-α and IL-8 in vitro and in vivo, and QTF could decrease TNF-α and IL-8 levels. In addition, we also observed that MP infection increased the ROS generation, and this finding was consistent with Guoping Sun et al(15).

Accumulating researchers demonstrate that ER stress and sustained UPR signaling contribute a lot to viral infections and inflammatory disorders(16). Viruses may interact with the host UPR and the release of virions generate abundant unfolded or misfolded proteins causing ER stress(17). Chlamydia pneumoniae infection has been found to induced ER stress/UPR, leading to the increased levels of ROS and cytosolic Ca²⁺(18). It is well documented that ER stress is strongly associated with pneumonia, such as COVID-19(7). Upregulation of eIF2 phosphorylation, ATF4, and CHOP are observed after SARS-CoV infection, resulting the activation of PERK(19). In the current study, PERK signaling pathway was activated by MP infection, and QTF could block that, suggesting QTF reduced MP infection-induced ER stress via PERK signaling pathway. Moreover, the activation of the NF-κB pathway caused the infection of many bacterial pathogens through induction of the host UPR has been well studied(20, 21). We also found the activation of the NF-κB after infection of MP in this study, suggesting that MP enhanced the NF-κB pathway through increase of the ER stress by upregulating PERK.

QTF alleviated MP infection-induced cytokine release, ROS production, and ER stress via inhibiting PERK signaling pathway.

Funding

This article is supported by National Natural Science Foundation of China (81804144 and 81674024).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

Zhiyan Jiang contributed to the concept, design, and manuscript review; Xiuxiu Liu and Mingjing Wang contributed to the experimental studies, manuscript preparation, and manuscript editing. Qianna Kan and Lin Yan contributed to experimental studies, data acquisition, and statistical analysis.

Ethics approval

Ethical approval for the study was provided by the independent ethics committee of Shanghai University of Traditional Chinese Medicine.

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No competing interests reported.

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Qingfei Tongluo formula mitigates Mycoplasma pneumoniae infection via PERK signaling pathway

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