Overexpression of Lias Gene Alleviates Pulmonary Injury Induced by fine particulate matter in Mice

doi:10.21203/rs.3.rs-1599359/v1

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Overexpression of Lias Gene Alleviates Pulmonary Injury Induced by fine particulate matter in Mice

https://doi.org/10.21203/rs.3.rs-1599359/v1

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Oxidative stress, inflammation is generally regarded as important toxicity mechanisms induced by fine particulate matter (PM_2.5). The antioxidant baselines of human body affect the intensity of oxidative stress in vivo. Our study aimed to evaluate the role of endogenous antioxidant capacity in PM_2.5-induced pulmonary injury using a novel mouse model (Lias^H/H) which approximately increased 150% endogenous antioxidant capacity relative to its wild-type counterpart. Lias^H/H and C57BL/6J mice were randomly divided into control group and PM_2.5 exposure group (n = 10) respectively. The PM_2.5-exposed group and the control group mice were intratracheally instilled with PM_2.5 and saline respectively once daily for 7 consecutive days. The main pulmonary pathological changes, oxidative stress and inflammatory biomarkers levels were examined. The results showed that oxidative stress was significantly increased in all mice after PM_2.5 exposure. Overexpression of Lias gene significantly increased the capacity against oxidative stress and decreased inflammatory responses caused by PM_2.5 in model mice compared with the control mice. Antioxidant capacity in the Lias^H/H mice was elevated through increased NRF2, the activation of ROS-p38MAPK- NRF2 signaling pathway. The novel mouse model is useful for clarifying the mechanisms of pulmonary injury induced by PM_2.5.

Fine particulate matter

oxidative stress

Lias

endogenous antioxidant capacity

antioxidant mouse model

pulmonary injury

Air pollution is a serious threat to human health. PM_2.5 exposure can cause serious health effects in the body, compared to larger particles in the atmospheric particulate matter. Numerous research studies have indicated a close relationship between PM_2.5 concentrations and the occurrence and mortality rates of respiratory illness (Habre et al., 2014; Mossman et al., 2007). A cohort study of 500,000 subjects found that with every increase in PM_2.5 concentration of -10 µg/m³, the total mortality, cardiovascular disease and respiratory disease increased by 4%, 6% and 8% respectively in the United States (Pope et al., 2004). Animal experimental evidence also shows that PM_2.5 can lead to respiratory inflammation, declining of lung function, increasing frequency in asthma and deterioration of chronic obstructive pulmonary disease (COPD) (Davel et al., 2012; G. Wang et al., 2013; Willers et al., 2013).

The mechanism of adverse health effects induced by PM_2.5 remains uncertain. Oxidative stress, in addition to local and systemic inflammation are generally considered to be key mechanisms of PM_2.5-mediated toxicities (Weichenthal et al.,2013; Willers et al., 2013). There is increasing evidence that oxidative stress, impaired antioxidant defense is related to the damage in human and animal cells caused by PM_2.5. Recent epidemiological studies have revealed that PM_2.5 may still cause health hazard at very low levels of exposure which is far below the specified air quality standards (Feng et al., 2016). The intensity of oxidative stress and the degree of body damage are also affected and restricted by the antioxidant system (Feng et al., 2016; Saklayen et al., 2010). Evidence shows that baseline antioxidation in different individuals was significantly different (Block et al., 2008).

α-Lipoic acid (LA) is a potent antioxidant, synthesized by lipoic acid synthase (LIAS) in mitochondria (Dörsam et al., 2016). Lipoic acid plays a significant role in the antioxidant system of body because it can scavenge a variety of free radicals and reduce other antioxidants with unique characteristics. For example, it can eliminate various types of free radicals in the body, enhance the antioxidant capacity through transcriptional regulation, and recycle other antioxidants, such as vitamins C and E, MitoQ10, and glutathione. Oxidation of lipoic acid can also be reduced and regenerated by enzymatic reaction (Ambrosi et al., 2016; Casciato et al., 2018). Exogenous lipoic acid, by virtue of its antioxidant effect, has been shown to be beneficial in many metabolic and vascular diseases. Although the pharmacological effects of lipoic acid have been explored in many studies, the importance of endogenous lipoic acid is largely unknown. In order to verify our conjecture and simulate antioxidant states of body, we used a novel mouse model (Lias^H/Hmice). The Lias^H/H mice exhibit 150% overexpression of the Lias gene, compared to the Lias gene expression in WT mice, determined based qRT-PCR and western blotting analyses. The novel model mouse exhibits high total plasma antioxidant capacity and lipid peroxidation induced by oxidative stress. Throughout all generations, the Lias^H/H mice did not exhibit any abnormal performance or behaviors. Remarkably, previous studies have repeatedly showed that both the increase of endogenous lipoic acid and the supplement of exogenous lipoic acid can alleviate the development of arteriosclerosis and pulmonary fibrosis ( Yi et al., 2006; Yi et al., 2010, Zhao et al., 2020). Therefore, we wanted to explore whether overexpression of Lias gene can lower oxidative stress and relieve inflammation induced by PM_2.5. Our aim was to first clarify whether the dose-response relationship occurs between the antioxidant/oxidative stress level and the injury degree of respiratory system, and then attempt to explore the potential molecular mechanisms induced by PM_2.5. The results of this study supply important experimental evidence for protecting susceptible populations.

2.1 Animal models

Lias^H/H mice were genetically engineered mice with C57BL/6J genetic background. Lias^H/H mice were generated as previously described (Xu et al., 2016; Zhao et al., 2020). The mice were established by modifying the base sequence of lipoic acid synthase (Lias) mRNA 3'- untranslated region. The expression of endogenous Lias gene was overexpressed to nearly 150% in the mouse (Xu et al., 2016). Accordingly, the expression of LIAS protein and antioxidant capacity in Lias^H/H mice were raised ( Xu et al., 2016). C57BL/6J mice were bought (China Vital River Animal Technology Co., Ltd.). All mice were free diet. The room temperature was 20 ~ 25 ℃, the relative humidity was 60% ~ 70% in animal house, and the alternating time of day and night was 12h / 12h. Animal care and use were in accordance with the guidelines of Animal Ethics Committee from Xinxiang Medical University (No. XYLL-2017086).

2.2 PM_2.5 collection, particle size and metal element analysis

The sampling location was at the roof of building with seven floors in Xinxiang Medical University (35°17 '11.17″ N, 113°55 '59.12″ E). The sampling of PM_2.5 was carried out between November 2018 to March 2019, using a quartz microfiber membrane for 24 h by an intelligent particulate sampler (TH-150 C Type III) using the methods of Li et al. (J. Li et al., 2021). Briefly, quartz microfiber membranes with filtered PM_2.5 were cut into 1 cm × 1 cm pieces. PM_2.5 was extracted by freeze drying method (J. Li et al., 2021). The metal elements in PM_2.5 were examined by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Fisher, USA). The size and morphology of PM_2.5 from part of the filter film were observed under scanning electron microscope (su8100, Japan).

2.3 Animal grouping and PM_2.5 exposure

20 male Lias^H/H mice and 20 male C57BL/6J mice were randomly divided into control and PM_2.5-exposed groups (n = 10 in each group). PM_2.5-exposed groups were exposed to 40µL solution containing PM_2.5(4 mg/kg body weight) with inhalable intratracheal instillation performed by reference in previously published study (Li J. et al., 2021,Jin Y. et al.,2017). And control groups were instilled instead of 40 µL sterile saline, once a day for a total of 7 consecutive days. The mice were sacrificed after being anesthetized with isoflurane. Blood samples were collected from the femoral artery. The right lung was ligated. Bronchoalveolar lavage fluid (BALF) was collected according to the reported method from our research team (J. Li et al., 2019). Briefly, the left lung was washed three times using 1mL PBS, the recovery rate of alveolar lavage fluid was above 80% for each mouse. The part of the right lung was fixed in 4% paraformaldehyde for 24 h to evaluate pathological changes. The plasma and lung samples were stored at -80℃ until further examination.

2.4 Lung histological examination

The lung tissue fixed with 4% paraformaldehyde were embedded in paraffin. The morphological changes of lung were observed by microscope with the method of paraffin embedding and H&E staining. Tissues were made as sections with 3 µm thick. Then they were processed by graded alcohol dehydration, cleared in xylene, mounted, and stained with H&E to evaluate the extent of pathological changes.

2.5 Assessment serum and lung tissue oxidative stress

Total antioxidant capacity (TAC) in the serum was examined using commercial kits. The levels of superoxide dismutase 2 (SOD2) and malondialdehyde (MDA) in lung tissue were examined with commercial kits following the published methods (Janero, 1990). The reactive oxygen species (ROS) in lung tissue were detected by DCF fluorescence (G. Xu et al., 2021). Fresh left lung tissue (20 mg) was placed in a glass homogenizer for full homogenization. The lung homogenate was diluted 100 times with PBS to prepare the cell suspensions. The levels of ROS in the different groups were measured using the DCFH-DA commercial kit. After 30 min incubation with DCFH-DA at 37°C, fluorescence intensity was measured, at the 525 nm emission wavelength, upon excitation at the 480 nm wavelength.

2.6 Serum and BALF cytokine measurement

The concentration of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in the BALF and serum of all mice were determined with commercial ELISA kits (Wuhan Huamei Biotechnology Co. Ltd, Wuhan, China).

2.7 Immunohistochemical method to analysis of NRF2 and P38 MAPK protein expression

Expression of NRF2 and P38MAPK protein in the lung was performed by immunohistochemical staining. The lung tissue sections embedded by paraffin were dewaxed with xylene and absolute ethanol, next incubated with the primary antibodies Nrf2 (1:100) and p38 MAPK (1:100) with the slices at 4°C overnight, respectively. In order to reduce non-specific background, the sample was pre-treated with hydrogen peroxide before incubation with HRP-conjugated antibody. The secondary antibodies were incubated for 50 min at 25°C. Then 3,3-diaminobenzidine (DAB) washed and incubated the sections for 15 min. Positive staining appeared in brown-yellow color. Images were observed and collected by a microscope. The intensity of the positive cell area was blindly quantified by Image software (version 6.0).

2.8 Assay of the genes in lung

Total RNA in Lias^H/H and C57BL/6J mouse lungs was extracted using Trizol (Thermo Fisher, USA). RNA concentrations were determined with a Nanodrop 1000 spectrophotometer (Thermo Fisher, USA). RNA was reverse transcribed to cDNA using the M-MLV kit (Thermo Fisher, USA). The expression of Nrf2, Keap1, HO-1, CAT, Lias and p38 MAPK mRNAs was tested using the SYBR qPCR Mix Kit (Promega). The cDNA primer sequences used were: Nrf2:5′-CAGCCATGACTGATTTAAGCAG-3′ and 5′-CAGCTGCTTGTTTTCGGT ATTA-3′; Keap1: 5′-ACTGGGTCAAATACGACTGC- 3′ and 5′-GAATATCTGCAC CAGGTAGTCC-3′; HO-1: 5′-GAAGACATCATTCAA CTACGCC-3′ and 5′-GAA GACATCATTCAACTACGCC-3′; CAT:5′-CACCTTCAAGTTGGTTAATGCA-3′ and 5′-CATGACCTGGATGTAA AACGTC-3′; Lias: 5'- ATG TAGAAA CAG TCC CGG AAT T -3' and 5'- TCT CGC CTA AAC CCA ACA TTAT-3'; p38MAPK:5′-ATCTCAACAAAGTTCGAGTTGC-3′ and 5′-GTCTGAAGCGCAGTA AGATTTT-3′. Amplification and detection of specific products were performed using the LightCycler® 96 (Roche Diagnostics, Mannheim, Germany). Cq values were generated by the Roche software, and fold changes were calculated with the 2^−ΔΔCq method.

2.9 Western blotting method to evaluate the expression of LIAS and some important proteins of the Nrf2 signal pathway

Total protein in the lung tissues or their mitochondria of each group were extracted. The lung mitochondria were separated from the homogenate by differential centrifugation using Tissue Mitochondria Isolation Kit and RIPA Lysis Buffer (Beyotime, China). Total protein was extracted from the lung mitochondria. The protein concentrations of lung tissues or mitochondria were determined with BCA Protein Assay Kit (Beyotime, China). Equal amount of total protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 µm polyvinylidene difluoride membrane. Membranes were blocked with 5% nonfat milk at 25°C, for 2 h. The membrane was incubated with the following primary antibodies: LIAS (1:1000, Proteintech, USA), KEAP1, Nrf2 (1:2000, Affinity Biosciences, USA), GCLC (1:2000, Abcam, USA) at 4°C overnight. The membrane was subsequently rinsed with TBS for three times and incubated with horseradish peroxidase combinative anti-rabbit secondary antibody (1:3000, Earthox, San Francisco, USA) for 2 hours. The protein bands were visualized by the Amersham ECL Reagent (GE Healthcare, USA). Membranes were incubated with either COXIV antibodies (1:3000, Proteintech, USA) to be the reference. The protein density bands were performed by ImageJ software.

2.10 Immunofluorescence analysis

After lung tissue sections were deparaffinized, they were incubated with anti-NRF2 antibodies (1:100; Affinity, USA) at 4°C overnight, and then incubated with Cy3-conjugated secondary antibodies (1:200; Affinity, USA) at 25°C and it kept for 50 min voiding light. Nuclei were stained with DAPI. Cells double-stained were observed by laser confocal microscopy. The NRF2 fluorescence intensity was evaluated with double blind method.

2.11 Statistical analysis

The data were analyzed using SPSS 22.0 software and expressed as mean ± SD. Normality of data was evaluated with the Shapiro-Wilkotest. The significant differences between the groups were compared by One-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. P < 0.05 was considered significant.

3.1 PM_2.5 metal element and particle size analysis

Table 1 shows 17 main types of water-soluble metal elements in some of the PM_2.5 which was intratracheally instilled to mice in this experiment. The order of the concentrations of these metal elements was: Zn > Fe > Al > Pb > B > Ge > Mn > Cu > As > Ba > Cd ¹¹²> Sr > Cd ¹¹⁴ > Cr > Pd > Ni > V. The concentrations of Zn, Fe, Al, and Pb were higher, at 809.79, 301.22, 297.65, and 234.71 ng/m³, respectively. The particle size and morphology of PM_2.5 were tested by scanning electron microscope. As show in Fig. 1A, most particles are less than 2.5µm in diameter and adhere to the fibers.

Table 1

PM_2.5 concentrations and elemental composition analysis
Elemental composition	Concentration (ng/m3)	Elemental composition	Concentration (ng/m3)
B	105.51 ± 28.20	Ge	60.86 ± 4.27
Al	297.65 ± 16. 04	As	39.17 ± 2.68
V	4.54 ± 0.19	Sr	15.97 ± 0.82
Cr	13.87 ± 1.65	Pd	13.42 ± 1.14
Mn	47.36 ± 5.37	Cd¹¹²	16.33 ± 1.50
Ni	5.39 ± 0.66	Cd¹¹⁴	15.54 ± 0.90
Cu	42.84 ± 2.45	Ba	29.25 ± 3.86
Zn	809.79 ± 26.61	Pb	234.71 ± 60.98
Fe	301.22 ± 2.45

3.2 High expression of Lias gene reduces the lung tissue damage in the Lias^H/H mice exposed to PM_2.5.

Herein, we used the Lias^H/H mice, the novel mouse models which exhibit 150% overexpression of the Lias gene compared to the Lias gene expression in WT mice, so as to investigate biological effects of increased endogenous antioxidant capacity on lung damage caused by PM_2.5. The Lias^H/H mice were established by modifying the base sequence of lipoic acid synthase (Lias) mRNA 3 '- untranslated region. (Fig. 1B). The expression of LIAS protein in the lung mitochondria and the total antioxidant capacity (TAC) in the serum were compared between the WT mice and the Lias^H/H mice. The levels of LIAS expression in lung mitochondria and TAC in the serum were both increased in Lias^H/H mice, as expected (Fig. 1C, D, E). As shown in Fig. 1E, the pulmonary histological alterations of the control and Lias^H/H mice were observed after exposure to PM_2.5, as well as the H&E-stained pulmonary sections, revealed minor damage in the architecture of mouse lung tissues in the C57BL/6J and Lias^H/H mice control groups. The alveolar septum of C57BL/6J control mice exposed to PM_2.5 appeared significantly wider, accompanied with obvious inflammation and infiltrations of the lymphocytes and plasma cells. In contrast, the alveolar septum of Lias^H/H mice exposed to PM_2.5 was much narrower, and the infiltration of lymphocytes and plasma cells was significantly reduced, indicating that inflammation was attenuated (Fig. 1F). Collectly, these data suggest that increased Lias gene expression could mitigate pulmonary injury.

3.3 Overexpression of Lias gene decreased oxidative stress

To evaluate whether PM-induced oxidative stress was systematically decreased due to enhanced endogenous antioxidant activity, we measured plasma and lung antioxidant levels using multiple biomarkers. As shown in Fig. 2A-D, the levels of ROS and MDA in the lung were significantly increased, the levels of T-AOC in the plasma and SOD2 in the lung were significantly reduced in the C57BL/6J mice after PM_2.5 exposure (P < 0.05). However, plasma T-AOC, lung SOD2 and ROS levels in Lias^H/H mice did not significantly change after exposure to PM_2.5 (P < 0.05). There was minimal difference of MDA production between Lias^H/H and C57BL/6J mice after PM_2.5 exposure (Fig. 2D). To further investigate the effects of LA on Lias^H/H and C57BL/6J mice after exposure to PM_2.5, we evaluated the expression of LIAS protein in lung mitochondria using western blotting (Fig. 2E), and the quantitative assessment of the densitometry (Fig. 2F). The level of LIAS protein was decreased in all mice after they were exposed to PM_2.5. However, the decline in LIAS protein levels was mild in Lias^H/H mice compared with C57BL/6J mice. It suggested that overexpression of lipoic acid synthase could be partially offset the decline in LIAS protein levels and then maintenance of LIAS protein alleviated pulmonary injury induced by PM_2.5.

3.4 High endogenous antioxidant level attenuates inflammatory response

To determine whether mice with enhanced antioxidant capacity could reduce inflammatory responses, we next examined the concentrations of inflammatory factor (TNF-α, IL-1β, IL-6, MCP-1) in BALF and serum. The levels of BALF TNF-α, IL-1β, MCP-1 and IL-6 in the C57BL /6J mice exposed to PM_2.5 were significantly higher than those in their C57BL/6J littermates (P < 0.05) (Fig. 3A-D). Overexpression of the Lias gene significantly reduced the PM_2.5-induced increase of TNF-α, IL-1β and IL-6 levels in BALF. An obvious difference was seen in BALF TNF-α, IL-1β, and IL-6 levels between the C57BL/6J group and the Lias^H/H group, simultaneously exposed by PM_2.5 (P < 0.05) (Fig. 3A, 3B, 3D). However, no significant difference was seen in MCP-1 levels between the two PM_2.5-exposed groups (Fig. 3C). What's more, we examined the inflammatory factors level of TNF-α, IL-1β, IL-6, and MCP-1 in the serum. The TNF-α, IL-1β, IL-6, and MCP-1 levels in the serum of the C57BL/6J group exposed to PM_2.5 were markedly higher than those of C57BL/6J control group (P < 0.05) (Fig. 3E-H). The levels of TNF-α, IL-1β, IL-6 and MCP-1 in Lias^H/H mice were increased, but the alteration was smaller compared with that observed in PM_2.5 exposed C57BL/6J group. Taken together, these data implied that high endogenous antioxidant level attenuates inflammatory response in Lias^H/H mice.

Increased endogenous antioxidant activity increased Nrf2 levels and modulated the gene expression of the Nrf2 signal pathway

Transcription factor Nrf2 is closely related to endogenous antioxidant capacity. It regulates the expression of downstream detoxifying enzymes including SOD, HO-1, CAT and GCLC (Casciato et al.,2018;Zhao et al,2020). Nrf2 plays a major part in preventing toxicity caused by particular matters by protecting against oxidative damage and inflammation. (Huang et al., 2015, Michal et al., 2020). Figure 4A showed the NRF2 protein expression in lung tissues which was measured by immunohistochemical staining. The NRF2 protein expression level was increased in PM_2.5-exposed C57BL/6J mice (P < 0.05). Correspondingly, NRF2 protein baseline in the Lias^H/H mice was significantly increased. In contrast, the NRF2 protein expression level decreased after exposure to PM_2.5 (P < 0.05). The difference in NRF2 protein levels was significant between C57BL/6J mice and Lias^H/H mice after exposure to PM_2.5 (P < 0.05). Semi-quantitative analysis revealed significantly higher AOD (IOD SUM/Area SUM) in Lias^H/H mice compared with the C57BL /6J mice exposed to PM_2.5 (P < 0.05) (Fig. 4B). Moreover, the expression of NRF2 in the nucleus of mouse lung tissues was further examined by immunofluorescence analysis after PM_2.5 exposure. Compared with C57BL/6J mice, the fluorescence intensity in the nuclear expression of NRF2 in lung tissues was remarkably increased in Lias^H/H mice (Fig. 4C). This outcome was consistent with the immunohistochemistry results. NRF2 protein levels were increased in C57BL/6J mice after exposure to PM_2.5 (P < 0.05). The NRF2 protein level was high at first in Lias^H/H mice but largely diminished following PM_2.5 exposure (P < 0.05). The results demonstrated that overexpression of LIAS activated the expression of Nrf2 and increased antioxidant capacity in Lias^H/H mice, which could be exhausted by PM_2.5 exposure.

To further explore the mechanisms underlying the alleviation of pulmonary damage caused by PM_2.5 via upregulated endogenous antioxidant capacity, the expression of Nrf2, Keap1, HO-1, and CAT mRNA associated with the Nrf2 signaling pathway were evaluated. Exposure to PM_2.5 significantly increased Nrf2 gene expression in C57BL/6J mice (Fig. 5A), consistent with the immunohistochemical staining and immunofluorescence results. Compared with C57BL/6J mice, Nrf2 gene expression level was approximately 150% higher in Lias^H/H mice by RT-PCR (Fig. 5A). However, Nrf2 was drained in large quantities in Lias^H/H mice after exposure to PM_2.5 (P < 0.05). Compared with C57BL/6J mice, the Nrf2 level was significantly upregulated in Lias^H/H mice (P < 0.05). Activation of Nrf2 is closely related to KEAP1 protein. Normally, Nrf2 is bound to KEAP-1 in the cytosol. Activated Nrf2 in response to stress leads to releasing Nrf2 to translocate to the nucleus and detaches from Keap1. The levels of Keap1 mRNA were decreased in all mice after PM_2.5 exposure, but the decrease was much milder in Lias^H/H mice compared to C57BL/6J mice. The levels of HO-1 and CAT mRNA, antioxidant enzymes downstream of Nrf2, were increased in C57BL/6J mice after PM_2.5 exposure (P < 0.05). Levels of HO-1and CAT mRNA were higher in Lias^H/H mice but declined sharply after exposure to PM_2.5 (P < 0.05). Changes in Nrf2, Keap1, HO-1, and CAT mRNA were significant different between C57BL/6J and Lias^H/H mice after exposure to PM_2.5 (P < 0.05). In line with the alteration of LIAS protein levels, Lisa gene levels were higher in Lias^H/H mice than those in C57BL/6J mice (P < 0.05). Compared with C57BL/6J mice, the mRNA level of Lias was marginally dropped in Lias^H/H mice after exposure to PM_2.5 (P < 0.05). These data showed that overexpression of Lias gene promoted Nrf2 expression and modulated the Nrf2 signal pathway. In addition, we measured the protein expression of KEAP1, NRF2 and GCLC in lung tissues of Lias^H/H and C57BL/6J mice exposed to PM_2.5. In support of these results, western blotting exhibited that the levels of NRF2 protein were visibly higher in Lias^H/H mice than that in C57BL/6J mice. The expression levels of NRF2 protein were increased in PM_2.5- exposed C57BL/6J mice, in contrast, the NRF2 protein expression level in Lias^H/H mice was decreased after exposure to PM_2.5 (P < 0.05) (Fig. 5B,5C). The KEAP1 protein expression level was decreased in C57BL/6J mice and Lias^H/H mice after PM_2.5 exposure (P < 0.05) (Fig. 5B-D). The change of GCLC protein levels was consistent with that of NRF2 in C57BL/6J mice and Lias^H/H mice after PM_2.5 exposure (P < 0.05). Therefore, we concluded that high endogenous antioxidant activity could decrease oxidative stress and strengthen antioxidant defense, likely due to an increase in Nrf2 and LIAS production, which modulated the antioxidant enzymes gene expression of the Nrf2 signal pathway.

3.6 Upregulation of antioxidant activity affected p38MAPK protein expression

In addition, we evaluated the effect of overexpression of the Lias gene on the p38MAPK protein. Figure 6A and 6B show p38MAPK protein expression in lung tissues was examined by immunohistochemical staining and RT-PCR, respectively. The results exhibited that exposure to PM_2.5 significantly increased the protein and gene expression level of p38MAPK in C57BL/6J and Lias^H/H mice (P < 0.05). The p38MAPK protein and mRNA levels showed a mild decrease in Lias^H/H mice than those in C57BL/6J mice after PM_2.5 exposure.

Documented studies have shown that the antioxidant baseline varies widely in different populations (Kakkar et al., 1997). We hypothesized that the basic endogenous antioxidant levels in the body may affect lung toxicity caused by PM_2.5. Therefore, in the present study, we investigated lung damage caused by PM_2.5, utilizing a novel mouse model simulating different antioxidant states in the human body. The results indicated that overexpression of Lias gene alleviates lung damage caused by PM_2.5 in mice possible through the strengthening of the antioxidant defense (including increased NRF2 and antioxidant genes and enzymes associated with Nrf2), resulting in decrease in oxidative stress and subsequent alleviation of the inflammatory response. It was assumed that the alteration to the endogenous antioxidant levels did not exceed a physiological range. The peak antioxidant level can reach 200% higher compared with the baseline if a healthy subject was given a pharmaceutical dose of LA at 600 mg (J. Chen et al., 2005). In this context, upregulation by ~ 50% of antioxidant level does not reach the pharmaceutical level. Therefore, these results showed the Lias^H/H mice can be administered as a precious animal model to expound the antioxidant role in pulmonary toxicity induced by PM_2.5.

Previous research has been confirmed that the toxicity of PM_2.5 depends not only on the concentration, but also on its complex chemical composition (Jesus A et al.,2009).

The highest concentrations in PM_2.5 were Zn, Fe, Al, Pb and they were respectively 809.79, 301.22, 297.65 and 234.71 ng/m³ in this study. The diameters of particles were less than 2.5µm and adhere to the fibers under the scanning electron microscope. The metal elements in the PM_2.5 samples of this study were consistent with those in a previous finding( L. C. Chen et al., 2009). Numerous studies demonstrate that PM_2.5 pollution is related to heavy metal components that partially mediates the adverse effects on the respiratory system. Exposure to zinc oxide or soluble zinc salt in environment can seriously affect health in inducing mitochondrial dysfunction, increasing in ROS and inflammation (Wu W., et al.,2013). Iron contributes to the most the generation of critical redox ROS through Fenton-type reactions (Charrier, J. G., et al.,2011, Di Pietro et al.,2011). Exposure to Zn, As, Cd, Hg, and Pb was related to respiratory dysfunction (reduced FVC and FEV1) and lung diseases, asthma, chronic obstructive pulmonary disease (COPD), bronchitis (L. C. Chen et al., 2009). The underlying mechanisms may involve metal-related activity of PM_2.5, i.e., the production of ROS, and oxidative stress (L. C. Chen et al., 2009, Riediker et al.,2019). It has been suggested that the ongoing studies aimed at elucidating adverse effects on the lung is related to the metals from PM_2.5.

We observed that there was minor lung injury in Lias^H/H mice after exposure to PM_2.5 than C57BL/6J mice, indicating the role of endogenous antioxidant level in pulmonary injury induced by PM_2.5. Although the mechanisms of PM_2.5-induced toxicity is uncertain (Feng et al., 2016), oxidative stress and inflammation have been considered to be major mechanism (X. Deng et al.,2013a; Weichenthal et al., 2013). Oxidative stress is the homeostatic imbalance of oxidative and antioxidant systems in cells with an excessive production of reactive oxygen species (ROS) (L. Wang et al., 2021). PM exposure can produce excessive ROS, causing oxidative stress and resulting in many biological processes such as inflammation or even the death of cells

(Riediker et al.,2019). PM stimulates mitochondria, resulting in the production of large amounts of ROS in mitochondria. Mitochondria is proposed to be cellular targets and plays an important role in PM_2.5-induced toxicity (Jin X, 2018). Further, evidence indicates that the antioxidant capacity of reproductive organs dropped significantly after exposure to PM_2.5, causing an oxidative stress response (Longhin et al., 2013; Torres-Ramos et al., 2011). We confirmed significantly higher antioxidant levels and lower oxidative stress in Lias^H/H mice. Thus, by using this model, we examined whether enhanced endogenous antioxidant defense in Lias^H/H mice can effectively suppress oxidative stress and consequently attenuate lung damage induced by PM_2.5. Although PM_2.5 exposure obviously increased the ROS and MDA levels in the lungs in the C57BL/6J mice, this effect was markedly reduced in Lias^H/H mice, likely due to higher levels of body antioxidant activity. Our results show that serum T-AOC and lung SOD concentrations were higher in Lias^H/H mice. Exposure to PM_2.5 significantly reduced T-AOC and SOD levels in C57BL/6J mice but this effect was milder in Lias^H/H mice after the two strains were exposed to PM_2.5. T-AOC reflects the total levels of various antioxidant substances in body, such as antioxidant macromolecules, small molecules, and enzymes. SOD is an important antioxidant enzyme that can effectively clean superoxide radicals and mediate the balance between oxidation and antioxidation (Deng et al.,2013b). These results strongly suggest that the basic endogenous antioxidant levels of the body are important for the effect of the pulmonary toxicity induced by PM_2.5 (Janero, 1990).

Inflammatory injury has been proposed as another main mechanisms induced by PM_2.5(J. Li et al., 2019). Elevated levels of pro-inflammatory cytokines and chemokines (IL-1β, TNF-α, and MCP-1) were found in the lung injury caused by PM_2.5 (He et al., 2017; J. Li et al., 2019). Similarly, this study also showed PM_2.5 obviously increased the levels of IL-1β, TNF-α, MCP-1 and IL-6 in C57BL/6J mice. Notably, the inflammatory response to PM_2.5 was reduced in Lias^H/H mice. This suggests that elevated endogenous antioxidant ability facilitates suppression of inflammation and subsequent pulmonary toxicity induced by PM_2.5.

Nrf2 as a crucial mediator of antioxidant response inside the cells, plays critical role in improving endogenous antioxidant capacity. Nrf2 regulates expression of downstream detoxifying enzymes including SOD, HO-1, CAT, and GCLC and facilitates the scavenging of excess ROS to protect against oxidative damage (B. Chen et al., 2015). Our data showed that the Nrf2 mRNA expression level was upregulated in C57BL/6J mice exposed to PM_2.5 compared with C57BL/6J control mice. Intriguingly, the initial high expression of the Nrf2 gene in Lias^H/H mice was declined following exposure to PM_2.5. Evidence showed that the level of Nrf2 is related to the time and dose of PM exposure. Acute exposure usually leads to an increase in Nrf2, while repeated exposure reduces Nrf2, and the levels of phase II detoxifying enzymes decreased (Michal et al.2020). The evidence supports our study, showing that acute exposure to PM_2.5 can increase Nrf2 level. Overexpression of Lias gene significantly upregulates endogenous antioxidant capacity and offsets the increased ROS in body.

ROS induced by PM_2.5 may function as signaling molecules to trigger the translocation of Nrf2 into the nucleus, resulting in altered transcription of the downstream antioxidant enzyme system (X. Deng et al., 2013a). Nrf2 regulates the antioxidant defense system, and it is limited and regulated by Keap1. When the antioxidant defense system of body is triggered, Nrf2 is activated and released from Keap1, next transferred into the nucleus, and regulates the expression of related antioxidant enzymes. A previous study showed that LA could promote Nrf2 translocation to the nucleus and upregulate eNOS and SOD1 activity, and activate phase II enzymatic detoxification by upregulating Akt phosphorylation (Shay et al., 2009). Therefore, we assumed that the alleviated PM_2.5-mediated pulmonary toxicity was due to increasing the expression of the Nrf2 upregulated by LA. Indeed, the results from this study showed that overexpression of Lias gene obviously upregulated the Nrf2 expression level and decreased Keap1 expression level in the lungs of Lias^H/H mice, which was supported by a previous study ( Sena et al., 2018). Higher expression of Lias gene potently raised the levels of Nrf2, HO-1, CAT and GCLC, although significantly reduced after PM_2.5 exposure. We assumed that LA may act on Keap1 / Nrf2 pathway, inhibit the binding of Keap1 and Nrf2, promote nuclear expression of Nrf2, cause body have high antioxidant capacity, and make the cells have a certain ability to resist oxidative damage when facing the attack of ROS and attenuate pulmonary injury induced by PM_2.5.

Studies have demonstrated that ROS-MAPK-Nrf2 signaling pathway is closely relevant to the adverse health effects caused by PM_2.5 (Liu etal.,2019). In order to furtherly explore the possible molecular mechanisms induced by PM_2.5 exposure, we measured the expression of p38MAPK mRNA and protein. p38MAPK is an important member of the MAPK family which are important transmitters of extracellular signals from the surface to the nucleus of cell and can be activated by oxidative stress and various proinflammatory factors. Evidence shows p38MAPK plays a significant role in the inflammatory response and anti-inflammatory response in cells (L. Li et al., 2015, Song et al., 2020). PM_2.5 exposure activated the ROS-mediated MAPK pathway, and then activates Nrf2 signaling pathway and induces apoptosis (Liu et al., 2019), resulting in antioxidant capacity reduced markedly in cell. PM_2.5 could significantly upregulate the expression levels of p38MAPK protein, as PM_2.5 exposure stimulates the production of excessive ROS (Cui et al., 2020). Upon ROS stimulation, p38MAPK phosphorylates Nrf2, separates it from Keap1 in the cytoplasm and aids in the translocation of Nrf2 into the nucleus as the decreasing of Keap1. Finally, it affects the antioxidant enzymes downstream of the Nrf2 signaling pathway, such as SOD, CAT and GCLC to protect tissues from oxidant injury (L. Li et al., 2015). The evidence was further supported our study, demonstrating that the expression of the p38MAPK gene and protein were increased after PM_2.5 exposure. However, the alteration of p38MAPK level was smaller in Lias^H/H mice than that of C57BL/6J group after PM_2.5 exposure. It suggested that overexpression of Lias gene in Lias^H/H mice could reduce inflammatory cytokine production and offset the adverse effects caused by PM_2.5 exposure through oxidative stress and activation of ROS-p38MAPK-Nrf2 signaling pathway.

In this study, we found that overexpression of endogenous Lias gene alleviates pulmonary injury induced by PM_2.5, likely through both increased LIAS, NRF2 levels and the activation of ROS-p38MAPK-Nrf2 signaling pathway. The protection effect is potentially conferred by Lias gene overexpression in Lias^H/H mice.

BALF	Bronchoalveolar lavage fluid
COPD CAT	Cronic obstructive pulmonary disease Catalase
GSH	Reduced glutathione
GCLC	Glutamate-cysteine ligase
HO-1	Haemoxygenase-1
Keap1	Kelch-like ECH-associated protein 1
LA	α-lipoic acid
LIAS MAPK	Lipoic acid synthase Mitogen-activated protein kinase
MDA	Malondialdehyde
MCP-1	Monocyte chemoattractant protein-1
Nrf2	Nuclear factor erythroid 2-related factor 2
SOD2	Superoxide dismutase 2
ROS	Reactive oxygen species
TAOC	Total anti-oxidation capacity
TNF-α IL-1β IL-6	Tumor necrosis factor-α Interleukin-1β Interleukin-6

Ethical Approval

Animal care and use were in accordance with the guidelines of Animal Ethics Committee from Xinxiang Medical University (No. XYLL-2017086).

Consent to Participate

The subjects were mice in this study.

Statements & Declarations

The authors declare no conflicts of interest relevant to this article.

Consent to Publish

All authors consent to publish.

Authors Contributions:

Guangcui Xu and Weidong Wu designed this project and writing draft. Guangcui Xu, Yingjun Tao, Cheng Xiong, Qiyu Gao completed the experimental work. Yingzheng Zhao, Zhen An performed software work and data curation. Mengdi LV, Fengquan Zhang did animal house work.

Funding

This research was funded by the Nature Science Foundation of Henan Provincial (grant 222300420516), the National Natural Science Foundation of China (grant 81703183, 81773399 and 81573112), International cooperation and exchange project of NSFC (grant 81961128031), Scientific and Technological Research Project of Henan Provincial (grant 222102320325 and 212102310643).

Competing Interests

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

Availability of data and materials

If the data is not suitable for disclosure, you can contact the corresponding author.

Acknowledgements

We are very grateful to the reviewers for giving us guidance to improve the quality of the manuscript.

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Overexpression of Lias Gene Alleviates Pulmonary Injury Induced by fine particulate matter in Mice

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Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Animal models

2.2 PM_2.5 collection, particle size and metal element analysis

2.3 Animal grouping and PM_2.5 exposure

2.4 Lung histological examination

2.5 Assessment serum and lung tissue oxidative stress

2.6 Serum and BALF cytokine measurement

2.7 Immunohistochemical method to analysis of NRF2 and P38 MAPK protein expression

2.8 Assay of the genes in lung

2.10 Immunofluorescence analysis

3. Results

3.1 PM_2.5 metal element and particle size analysis

3.3 Overexpression of Lias gene decreased oxidative stress

3.4 High endogenous antioxidant level attenuates inflammatory response

3.6 Upregulation of antioxidant activity affected p38MAPK protein expression

4. Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

Overexpression of Lias Gene Alleviates Pulmonary Injury Induced by fine particulate matter in Mice

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Animal models

2.2 PM2.5 collection, particle size and metal element analysis

2.3 Animal grouping and PM2.5 exposure

2.4 Lung histological examination

2.5 Assessment serum and lung tissue oxidative stress

2.6 Serum and BALF cytokine measurement

2.7 Immunohistochemical method to analysis of NRF2 and P38 MAPK protein expression

2.8 Assay of the genes in lung

2.10 Immunofluorescence analysis

3. Results

3.1 PM2.5 metal element and particle size analysis

3.3 Overexpression of Lias gene decreased oxidative stress

3.4 High endogenous antioxidant level attenuates inflammatory response

3.6 Upregulation of antioxidant activity affected p38MAPK protein expression

4. Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1

2.2 PM_2.5 collection, particle size and metal element analysis

2.3 Animal grouping and PM_2.5 exposure

3.1 PM_2.5 metal element and particle size analysis