Qingfei Tongluo Formula Mitigates Mycoplasma pneumoniae Infection via the PERK Signaling Pathway

Mycoplasma pneumoniae pneumonia (MPP) is usually found in school-aged children and relapses easily because of antibiotic resistance. The Qingfei Tongluo formula (QTF) is a clinically used traditional Chinese medicine to treat MPP. Our previous study demonstrated that QTF exhibited ameliorative effects on the experimental MPP mice model. In this study, the function and underlying QTF mechanism in MPP was attempted to be further explored. Mycoplasma pneumoniae (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 ER stress. MP infection was found to enhance cytokine release and ER stress in vitro and in vivo, and this effect 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 the PERK overexpression exhibited the opposite effects. In conclusion, QTF alleviated MP infection-induced cytokine release, ROS production, and ER stress via PERK signaling pathway inhibition.


Introduction
Respiratory tract infection has been, and remains to be, a major public health concern, which can range in severity from mild to life-threatening, and is especially dangerous in children [1]. Mycoplasma pneumoniae (MP), a common bacterial pathogen, is considered as an important respiratory tract infection cause [2]. MP is also associated with community-based pneumonia, particularly among schoolaged children and young adults. MP-induced pneumonia (MPP) easily relapses due to antibiotic resistance. Therefore, new and more effective drugs are needed. Traditional Chinese medicine attracted the researchers' attention due to its increased safety and effectiveness.
The endoplasmic reticulum (ER) is an important organelle in eukaryotic cells, which is responsible for the synthesis and folding of proteins and storage of free calcium [7]. Some physiological stress including Ca 2+ levels, disturbances in redox, or other environmental factors induce misfolded protein accumulation leading to ER stress. Triggering the unfolded protein response (UPR), the ER stress mainly involves three signaling pathways: inositol-requiring enzyme-1α (IRE1α), activating transcription factor 6 (ATF6), and protein kinase RNA-like endoplasmic reticulum kinase (PERK) [8]. UPR is also associated with several major cellular activities including proinflammatory response, autophagy, apoptosis, innate immunity, and the mitogen-activated protein kinase pathways [9].
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. For MP infection, the culture medium was replaced with MP infection medium containing 10% MP (approximately 1 × 10 7 CFU/10 6 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.

Methods
2.3. ELISA. The ELISA kit (Jiancheng, Nanjing, China) was used to determine the IL-8 and TNF-α contents according to the manufacturer's protocol.

MP Infection Induces Cytokine Expression and ER Stress.
MP was applied to A549 cells and cultured for 12, 24, and 48 h. MP infection increased the production of TNF-α and IL-8 in a time-dependent manner as shown in Figure 1(a). ATF6, PERK, and IRE1α are the three major signaling pathways involved in ER stress. Therefore, the levels of ATF6, PERK, and IRE1α were detected using real-time PCR. The results illustrated significantly upregulated PERK and ATF6 mRNA levels upon MP infection. However, there  Disease Markers was no significant change in IRE1α level (Figure 1(b)), indicating that IRE1α signaling pathway may not be affected by MP infection. ELISA assay showed that the IL-8 and TNF-α increase induced by MP was inhibited after MP treatment in a dose-dependent manner (Figure 2(a)). Nuclear factor-κB (NF-κB) is a common anti-inflammatory target that has been related to proinflammatory cytokine production, such as TNF-α and IL-8 [10]. The nuclear NF-κB level was promoted after MP infection as shown in Figure 2(b) but depressed upon QTF treatment. Opposite effects were observed of the cytoplasmic NF-κB level, indicating that MP induced the NF-κB activation which could be inhibited by QTF. Furthermore, ROS has been reported to regulate NF-κB activity, so we detected ROS level upon treatment with MP and QTF. MP infection increased the ROS level in A549 cells as expected; however, cells treated with MP and QTF exhibited lower ROS level than the cells treated with MP only (Figure 2(c)). In addition, the increase PERK and ATF6 levels induced by MP were alleviated after QTF treatment (Figure 2(d)), suggesting that PERK and ATF6 signaling pathways were involved in QTF function in MPtreated cells. MP infection upregulated the protein levels of PERK, p-eIF2α, ATF4, and CHOP as shown in Figure 2(e). QTF could decrease these protein levels in a dosedependent manner. The TUNEL results showed that MP infection increased the apoptotic cell number, and QTF reduced the apoptotic cell number (Figure 2(f)). MP infection enhanced caspase 3, CHOP, and PERK expression   Disease Markers according to the immunofluorescence (Figure 2(g)), while QTF alleviated these effects. Taken together, QTF mitigated the effects induced by MP infection in A549 cells.

PERK Silencing Alleviates MP Infection-Induced
Cytokine Release, ROS Production, and ER Stress in A549 Cells. Knockdown lentiviruses targeting PERK were applied to A549 cells upon MP infection to confirm whether PERK was involved in MP infection. The three shPERK lentiviruses significantly decreased the protein PERK level in A549 cells as shown in Figure 3(a). The shPERK-2 gives the best silenc-ing effect. Thus, shPERK-2 lentivirus was used in the following studies to knockdown PERK level. Furthermore, this study found that PERK silencing could alleviate MP infection-induced TNF-α and IL-8 release (Figure 3(b)), NF-κB activation (Figure 3(c)), and ROS production (Figure 3(d)). In addition, the PERK, p-eIF2α, ATF4, and CHOP level increases induced by MP infection were inhibited by PERK silencing (Figure 3(e)). TUNEL results showed that PERK silencing reduced the increased apoptotic cell number induced by MP infection (Figure 3(f)). According to the immunofluorescence (Figure 3(g)), MP infection

QTF Alleviates PERK Overexpression-Induced Cytokine
Release, ROS Production, and ER Stress in A549 Cells. PERK overexpressing lentivirus was used to enhance the PERK expression. As expected, the PERK protein level was markedly upregulated by PERK overexpressing lentivirus (Figure 4(a)).
ShPERK depressed MP infection-induced activation of NF-κB based on the results in Figure 3(c). PDTC, an NF-κB inhibitor, was applied to A549 cell. The PERK overexpression promoted the TNF-α and IL-8 release as shown in Figure 4(b); however, cotreatment with PERK overexpressing lentivirus and PDTC showed lower TNF-α and IL-8 levels than that in the cells treated with PERK overexpressing lentivirus alone. Moreover, the PERK overexpression could increase the TNF-α and IL-8 release (Figure 4(c)), NF-κB activation (Figure 4(d)), ROS production (Figure 4(e)), and The TUNEL results showed that the PERK overexpression induced apoptotic cell increase, and that QTF could reduce the apoptotic cell number (Figure 4(g)). QTF could decrease the enhanced caspase 3, CHOP, and PERK expressions induced by the PERK overexpression. In summary, these results indicated that QTF played a protective role in the A549 cell via PERK signaling pathways.

QTF Mitigates MP Infection-Induced Cytokine Release and ER Stress In Vivo.
The MP infection mouse model was established and treated with low, middle, and high dose of QTF to further explore QTF function. HE staining showed that relatively normal lung tissues were observed in the control group. There were many pulmonary interstitial infiltrations of lymphocytes and plasmacytes, as well as bronchus and vasodilation congestion in the model group. QTF relieved the injury induced by MP infection in a dosedependent manner ( Figure 5(a)). Furthermore, this study found that QTF alleviated MP infection which induced the release of TNF-α and IL-8 ( Figure 5(b)), NF-κB activation ( Figure 5(c)), and the protein levels of PERK, p-eIF2α, ATF4, and CHOP ( Figure 5(d)) in vivo, indicating that QTF played a protective role in the MP infection mouse model.

Discussion
MP infection is the cause of several diseases including pneumonia. The MPP incidence in children is increasing [11]. Researchers prefer to focus on exploring the mechanism under antibiotic MPP treatment. The effects of traditional Chinese medicine were usually ignored. QTF was initially developed by the Pediatric Department at Shanghai Longhua Hospital and exhibited an effective role in the treatment of children with MPP [12]. Our previous study found that QTF showed ameliorative effects on the MPP mice model [3]. This study demonstrated that QTF alleviated MP infection-induced cytokine release, ROS production, and ER stress in vitro and in vivo. When infection occurs, our body's first response should be to the inflammation caused by the innate immunity.
TNF-α and IL-8 are the two important cytokines of the inflammation process. TNF-α, which is produced by monocytes and macrophages, plays a key role in MP infectioninduced lung injury [13]. Higher TNF-α serum level is observed in children with MPP [14]. Recently, high expressions of both TNF-α and community-acquired respiratory distress syndrome toxins are considered to be a good predictor for MPP [15]. MP has been found to induce IL-8 production in bronchial epithelial cells via NF-κB or ERK signaling pathway [16]. This current study found that MP infection enhanced the TNF-α and IL-8 release in vitro and in vivo, and QTF could decrease TNF-α and IL-8 levels. In addition, MP infection was also observed to increase ROS generation, and this finding was consistent with Sun et al. [17].
A growing number of researchers demonstrate that ER stress and sustained UPR signaling contribute significantly to viral infections and inflammatory disorders [18]. Viruses may interact with the host UPR and the release of virions that generate abundant unfolded or misfolded proteins causing ER stress [19]. Chlamydia pneumoniae infection has been found to induce ER stress/UPR, leading to increased ROS and cytosolic Ca 2+ levels [20]. It is well documented that ER stress is strongly associated with pneumonia, such as COVID-19 [9]. EIF2 phosphorylation, ATF4, and CHOP upregulation are observed after SARS-CoV infection, resulting in PERK activation [21]. In the current study, PERK signaling pathway was activated by MP infection, and QTF could block that, suggesting that QTF reduced MP infection-induced ER stress via PERK signaling pathways. Moreover, the NF-κB pathway activation caused by the infection of many bacterial pathogens through induction of the host UPR has been well studied [22,23]. NF-κB activation after MP infection in this study was also found, suggesting that MP enhanced the NF-κB pathway through increase of the ER stress by upregulating PERK.

Data Availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
The authors have no relevant financial or nonfinancial interests to disclose.