Introduction

Mycoplasma pneumoniae (MPP) is the commonest cause of pneumonia in neonates (Gao et al. 2013). As per the WHO, 19% of under-five mortality is attributed to MPP (Guo et al. 2015). MPP is an inflammatory condition of alveoli in the lungs. It is frequently caused by viruses or bacteria and rarely by other microorganisms, drugs, and autoimmune diseases conditions (Shangguan et al. 2014). Bacterial pneumonia is treated with antibiotics. MPP is resistant to antibiotics and is difficult to cure (Ferrer et al. 2016). Moreover, MPP can also cause severe complications, such as asthma and pulmonary fibrosis (Wood et al. 2017).

Studies have suggested that pneumonia symptoms caused by MP are due to the initiation of pro-inflammatory cytokine levels (Shimizu 2016). MPP stimulates immune responses to kill the pathogens by upregulation of the cytokines such as IL-10, IL-8, TNF-α, and IL-6 and increasing leucocytes and neutrophils at the site of infection, ultimately leading to lung impairment (Wang et al. 2014). Severity of lung disease caused by MPP is associated with the immune response of the host (Waites et al. 2014). In general, corticosteroids are used for treating MPP to reduce the inflammatory response and pulmonary injury. However, corticosteroids cause various side effects. Thus, natural, safer and effective compounds are required for MPP treatment (Shangguan et al. 2014).

Sinomenine (SIN) is sequestered from the Chinese herb Sinomenium acutum (Bordon et al. 2013). SIN has anti-arrhythmic, anti-analgesic, anti-arthritic, apoptotic, and anti-inflammatory properties. SIN also prevents lung cancer, breast cancer, liver cancer, and other tumors (Zhao et al. 2012; Gao et al. 2013). However, not much is known about the anti-inflammatory and apoptotic action of SIN on MPP. Therefore, we explored the anti-inflammatory and apoptotic efficacy of MPP in the mice model.

Materials and methods

Chemicals

Sinomenine (purity > 95%) and mycoplasma pneumoniae (MPP) culture were purchased from ATCC, USA. Antibodies IL-6, TGF, IL-1, IL-8, and TNF-α were procured from Thermofischer Scientific, USA. Antibiotics, and all the other biochemicals were procured from Merck, Germany.

Mycoplasma pneumoniae (MPP) culture

MP strain ATCC15531 was cultured in Hayflick adapted medium comprising of PPLO broth, horse serum (25%), penicillin-G, yeast extract, glucose (0.5%), Thallium acetate (0.025%), and phenol red (0.002%). The cells were cultured at 37 °C for 1 week (Lavazza et al. 2022).

Animal maintenance

BALB/c male mice aged 3-week-old weighing 15 ± 1 gm was procured from the departmental animal house and preserved in a controlled temperature, humidity and kept in aseptic cage dust pathogen free environment. All mice were acclimatized for 1 week before experiments. The research was approved by Xi'an Children's Hospital animal ethical committee (Approval No: XCH202106726A).

Experimental design

Mice were divided into 4 groups with 6 numbers each (n = 6/group).

Group-I mice were considered as control (normal).

Group-II mice were induced with pneumonia through 100 μl MP (1 × 107 ccu/ml). MP was administered through nasal drops for 2 days.

Group-III mice were administered SIN (20 mg/kg bw) after 3 days of successively giving MPP. MPP was administered through nasal drops for 2 days.

Group-IV mice were treated with SIN (20 mg/kg bw) alone for 3 days.

Subsequently, all the mice were sacrificed at the end of the experiment. Samples were collected for further biochemical and histopathological analysis.

Broncho alveolar lavage fluid (BALF) isolation

Five separate diluents of 30 mL buffered saline (0.89%) were instilled in the right side of central lobe of lung by inserting a catheter in the trachea of terminally anesthetized mice. The instilled fluid was gently retracted and the pooled fluid was rapidly centrifuged for 10 min at 6000 rpm. The clear solution was transferred to tubes and stored at 75 °C for subsequent testing as per the Van Hoecke et al. 2017 method.

Estimation of complete protein in BALF

The total protein in the BALF was assessed in normal and experimental mice using a kit for protein assay based on the Bradford method. BALF protein was considered as a marker of lung penetrability and epithelial injury (Caraballo et al. 2013).

BALF—total cell count

Draining method was used to separate the slime from BALF. Total cells were counted using a hemocytometer with a 5 mL dilution of BALF. The cells were counted and the percentages were calculated using the Zhang et al. 2020 method.

Analysis of MP–IgM, MCP-1, and inflammatory cytokines by ELISA

MP–IgM in the lung tissues and MCP-1, cytokines (IL-6, TGF, IL-1, IL-8, and TNF-α) in the serum of the mice were determined using Kienast et al. 1996 method by ELISA kit (Cayman chemicals, USA) as per manufacturer’s protocol.

Detection of MP–DNA by qRT–PCR

The DNA samples were extracted from the left lobe of the lung using a kit from Thermo Fisher Scientific (Waltham, USA), as per the manufacturer’s instructions. The RT–PCR MP kit was used to detect MP–DNA (Thermo Fisher Scientific, Waltham, USA). As a fluorescent indicator for RT–PCR, SYBR green (Synergy Brands, Inc. (stock symbol)—dsDNA binding dye) was used as per Abdel Sater et al. 2022 method. This procedure was carried out using a qPCR apparatus (Bio-Rad, USA) in accordance with the manufacturer's instructions. The following were the primer sequences:

MP–DNA, F: 5'-CAGAAACACACACAGCAGCTATT-3' R: 5'-CACGTTGATCCGCAAAGGAAGT-3'; GAPDH, 5'-TCTCCCTCACAATTTCCATCCC-3' R: 5'-TTTTTGTGGGTGCAGCGAAC-3'

Histological analysis

On the fourth day, the inferior lobe of the right lung was insulated with 5 percent formaldehyde, fixed with paraffin, cut into sections and stained with hematoxylin and eosin (H & E) as per Zhou et al. 2017 method. After staining, tissues were examined for signs of lung injury and inflammation under a microscope.

Western blotting analysis

The removed lung tissues were washed twice with PBS before being placed on ice for 30 min with an ice cold lysis buffer containing 0.01 percent protease inhibitor (Lichtenstein 2015). The lysate was centrifuged at 13,000 rpm for 10 min at 4 °C. The resulting solution was then processed through SDS–PAGE (Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis) (10%) and transferred to PVDF–(Polyvinylidene fluoride) film. After that, 5% fat-free milk was used to block the membrane and added with specific primary antibodies for Bax, Bcl-2, ERK, JNK, and NF-κB. The β-actin was then used as a standard and treated with horseradish peroxidase-conjugated secondary antibodies. Enhanced chemiluminescence was used to see the membrane (ECL, Millipore).

Statistical analysis

Results were expressed as mean ± SD. Data were analyzed using one way Analysis of Variance (ANOVA) with the Dunnett’s post hoc test. All statistical analysis were performed using software SPSS version 18.0. P value < 0.05 was considered statistically significant.

Results

Effect of SIN on the protein in BALF

The protein level in BALF was significantly increased in the MPP-induced experimental mice significantly as compared to the control group (p < 0.05). SIN treatment significantly reduced (p < 0.05) the total protein levels in BALF in SIN + MPP group (p < 0.05). Protein levels in SIN alone groupwere significantly (p < 0.05) similar to protein levels in the control mice. Thus, SIN proved to have ameliorative effects (Fig. 1a).

Fig. 1
figure 1

a Effect of SIN on whole proteins and total cells in BALF of experimental MPP mice. Results were expressed as mean ± SD of six observations.*P < 0.05 indicates the values not sharing a common superscript letter differ significantly. b Effect of SIN on MP–IgM in lung tissue and MCP-I in serum were measured by ELISA. Results were expressed as mean ± SD of six observations.*P < 0.05 indicates the values not sharing a common superscript letter differ significantly. c Anti-MPP action of SIN on mice model. The MPP–DNA’s relative expression was measured by qRT–PCR. Results were expressed as mean ± SD of six observations. *P < 0.05 indicates the values not sharing a common superscript letter differ significantly

Full size image

Effect of SIN on the cells in BALF

There was a BALF were analyzed in the experimental mice, the significant increase in the cells in BALF of the MP-induced group as compared to control group (p < 0.05). SIN treatment reversed and significantly reduced the increased total cell count in the SIN + MPP group (p < 0.05). SIN alone group retained similar cell count as the control group (Fig. 1a).

Effect of SIN on MP–IgM and MCP-1

MP–IgM and MCP-1 in the MPP-infected mice were significantly elevated in contrast to control mice. SIN significantly reversed the MP–IgM and MCP-1 levels in the MPP + SIN group (p < 0.05). The control group and SIN alone group showed similar values (Fig. 1b).

Effect of SIN on MPP

The relative expression of MP–DNA increased significantly in the MPP-group as compared to the control group (p < 0.05). SIN significantly reversed the augmented MPP–DNA expression in the MPP + SIN group. The MP–DNA expressions in control and SIN were similar (Fig. 1c).

Effect of SIN on inflammatory cytokines

The inflammatory cytokines play a dynamic role in the protection and inflammation reactions. The levels of cytokines (IL-6, TGF, IL-8, IL-1, and TNF-α) were significantly (p < 0.05) elevated in the MPP infected mice as compared to the control mice. However, SIN treatment significantly reduced (p < 0.05) these cytokines in the MPP + SIN group. The control and SIN alone mice had similar cytokines levels (Fig. 2).

Fig. 2
figure 2

Effect of SIN on the determination of pro-inflammatory cytokines in serum. Results were expressed as mean ± SD of six observations. *i < 0.05 indicates the values not sharing a common superscript letter differ significantly

Full size image

Effect of SIN on lung histopathology

Figure 3 depicts the lung histopathology in control and experimental mice. The MPP lung tissues of the mice displayed extreme damage to the tissues, increased inflammatory cells penetration, and vasodilation. Conversely, MPP + SIN treatment reversed the inflammation and tissue architecture towards normal. SIN-alone treated mice also preserved the normal tissue architecture.

Fig. 3
figure 3

Effect of SIN on the histopathology of lung tissues. A Control mice show normal lungs histology. B MPP infected mice show impairment of lung tissues, vasodilation, and inflammation. C MPP + SIN treated mice show lessened inflammation and reversal of the lung cells arrangements to normal D SIN alone treated mice display normal lungs architecture. (Stained with H&E, magnification X40)

Full size image

Effect of SIN on Bax/Bcl-2 protein expression

The control and SIN alone treated mice had reduced intensities of Bax and elevated intensities of Bcl-2. MPP-induced mice up-regulated the Bax expression and down-regulated Bcl-2. These findings indicated that SIN inhibits MPP-induced apoptosis via diminishing Bax and enhancing Bcl-2 levels (Fig. 4).

Fig. 4
figure 4

Effect of SIN on apoptosis by Bax/Bcl-2 protein expression. These protein expressions were analyzed by western blotting. Results were expressed as mean ± SD of six observations. *P < 0.05 indicates the values not sharing a common superscript letter differ significantly

Full size image

Effect of SIN on ERK/JNK/NF-κB protein expression

The effect of SIN on ERK, JNK, and NF-κB expressions was analyzed (Fig. 5). Protein expressions of ERK and NF-κB were significantly increased and JNK significantly decreased in the MPP group versus control group.

Fig. 5
figure 5

Effect of SIN on ERK/JNK/NF-kB signaling pathway. These protein expressions were analyzed by western blotting. Results were expressed as mean ± SD of six observations. *P < 0.05 indicates the values not sharing a common superscript letter differ significantly

Full size image

Discussion

MPP can cause damage to the respiratory tract, and other pulmonary problems. MPP causes acute bronchitis, acute asthma, and community-acquired pneumonia in children and adults (Xu et al. 2019). Multiple antibiotics have recently been found to be ineffective against MPP infection. MPP patients develop serious lung complications and require intensive care (Maselli et al. 2018). SIN is an isoquinoline that is being studied for the treatment of rheumatoid arthritis, inflammation, and several cancer models in the laboratory (Wood et al. 2017). The current study examines the effects of SIN on MPP in mice. The anti-inflammatory and anti-MPP mechanisms of action were also studied. Research suggests that MPP is linked to the cytokines expression and the initiation of pulmonic fibrosis (Zhu et al. 2016). MPP infection causes the production of cytokines, such as IL-6 and TNF-α, which excite many leukocytes congregated at the contamination site, resulting in respiratory impairment (Kurai et al. 2013). Furthermore, the critical inflammatory mediators IL-1 and TNF-α have been implicated in the advancement of primary inflammation, which can lead to the formation of lymphocytes and neutrophils (Lin et al. 2018). These MPP induced inflammatory mediators play a key role in the depletion of the alveolar medium (Boddeke et al. 2001). In the current investigation, SIN reduced the inflammatory mediators in serum, such as IL-8, IL-6, TNF-α, IL-1, TGF, and MCP-1. Thus, the study implies that SIN has anti-inflammatory properties (Wang et al. 2006). The NF-κB signaling pathway has been used to control inflammatory responses in the lungs (Wang et al. 2005). TNF-α, IL-1, IL-6, IL-8, and TGF are all required for the highest transcription of various cytokines (Wang et al. 2006). As a result, NF-κB inhibitors may be useful as an anti-inflammatory (Tornatore et al. 2012). MPP can be stopped by inhibiting phosphorylation of JNK and ERK. SIN has proven anti-inflammatory properties by inducing NF-κB (Liu et al. 2013). Our data also revealed that SIN inhibited JNK, ERK, and NF-κB phosphorylation. This implied that SIN is a useful forMPP treatment.

SIN inhibited the pulmonary damage through inhibiting MPP by apoptosis and pulmonic fibrosis protein degradation. The anti-apoptosis Bcl-2 family includes Bax and Bcl-2. These can inhibit caspase-3 induction and are implicated in apoptosis control (He et al. 2005). SIN has been shown as apoptosis regulator via Bax/Bcl2 expressions (Mu et al. 2016). Our findings also revealed that MP–DNA expression was higher in the lung tissues of MPP-infected mice. SIN treatment reversed the DNA expressions. MPP-induced mice had a high MP–IgM. These were significantly reduced with SIN treatment. The findings indicate that SIN inhibits MPP through the attenuation of inflammation via ERK/JNK/NF-κB signaling pathway.

Conclusions

Our findings reveal that MP–DNA expression was higher in the lung tissues of MPP-infected mice. SIN had inhibitory effect on MPP–DNA. MPP-induced mice had increased MP–IgM. SIN + MPP group had significant reduction in MPP–IgM levels indicating that SIN treatment inhibited MPP–IgM. These findings suggest that SIN inhibits MPP in mice by the suppression of the IgM reaction and attenuation of inflammation via ERK/JNK/NF-κB signaling pathway.