The Effect of Ganoderma Microsporum immunomodulatory proteins on alleviating PM2.5-induced inflammatory responses in pregnant rats and fine particulate matter-induced neurological damage in the offsprings

Fine particulate matter 2.5 (PM2.5) induces free radicals and oxidative stress in animals, leading to a range of illnesses. In this study, Ganoderma Microsporum immunomodulatory (GMI) proteins were administered to alleviate PM2.5-induced inflammatory responses in mother rats, and PM2.5-induced inflammatory responses and neurological damage in their offspring. The results suggested that GMI administration decreased the risk of neurological disorders in mother rats and their offspring by reducing the white blood cell count, lessening inflammatory responses and PM2.5-induced memory impairment, and preventing dendritic branches in the hippocampi from declining and microRNAs from PM2.5-induced modulation.

www.nature.com/scientificreports www.nature.com/scientificreports/ Measurement of oxidative stress cytokines level in serum. Serum oxidative stress cytokines were detected by Rat oxidative stress kit (Signosis EA-1501). First, add 100 μl standard and ten-fold diluted serum to each well for 2 hr at room temperature, then wash three times. Added Biotin labeled antibody 100 μl for 1 hr at  2.5 . *, and ** indicate p < 0.05, and p < 0.01 significant difference, respectively, compared with control. # , and ## show p < 0.05, and p < 0.01, respectively, compared with PM 2.5 -treated controls. Figure 2. GMI decreased oxidative stress induced cytokines. Oxidative stress cytokines in serum was detected by Rat oxidative stress kit, which shows that GMI is able to mitigate the oxidative stress induced by PM 2.5 exposure. *, and ** indicate p < 0.05, and p < 0.01 significant difference, respectively, compared with control. # , and ## show p < 0.05, and p < 0.01, respectively, compared with PM 2.5 -treated controls.
www.nature.com/scientificreports www.nature.com/scientificreports/ room temperature, then wash three times. And added Streptavidin HRP 100 μl at room temperature for 45 minutes. Finally read at 450 nm. (Process were followed Signosis Rat Oxidative Stress ELISA kit manual).

Measurement of IgE level in Serum.
IgE were detected by Anti-Ovalbumin IgE ELISA kit (Cayman, USA). First, add 100 μl standard and serum to each well for 2 hr at room temperature, then wash four times. Added ova-biotion conjugate 100 μl for 1 hr at room temperature, then wash four times. And added Streptavidin HRP 100 μl at room temperature for 30 minutes, then wash four times. Next add TMB for 30 min and HRP stop solution. Finally read at 450 nm. (Process were followed Cayman Anti-Ovalbumin IgE ELISA kit manual).

Measurement of Histamine level in Serum.
Histamine was detected by Histamine ELISA kit (Enzo, USA). First, add 100 μl standard and serum to each well for 1 hr at room temperature, and then wash three times. Added SA-HRP 200 μl for 30 mine at room temperature, then wash three times. Added TMB for 30 min and stop solution. Finally read at 450 nm. (Process were followed Enzo Histamine ELISA kit manual). . GMI reduce the PM 2.5 -caused inflammation in offsprings. (A) Leukocyte increased in offspring which maternal exposed to PM 2.5 , GMI decreased the level. (B) IgE (C) histamine level were restored in serum. *, and ** indicate p < 0.05, and p < 0.01 significant difference, respectively, compared with control. # , and ## show p < 0.05, and p < 0.01, respectively, compared with PM 2.5 -treated controls. (2019) 9:6854 | https://doi.org/10.1038/s41598-019-38810-5 www.nature.com/scientificreports www.nature.com/scientificreports/ Western Blot. Rats were euthanized via CO 2 asphyxiation, and the brains were removed. Tissues were homogenized in TEE buffer with PMSF, and used Triton X-100 to lysis cell membrane. Lysates were centrifuged (13000 rpm at 4 °C) for 10 min, and the supernatants were collected. Protein concentrations were determined by BCA assay kit. Proteins were loaded 50 μg/well on a 12% SDS-polyacrylamide gel and transferred to a PVDF membrane. Nonspecific reativity was blocked for 1 h at room temperature and primary anti-CD68, anti-catalase, anti-SOD-1 and anti-GAPDH were incubated overnight at 4 °C. Then, the membranes were washed for 20 min for three times. Secondary antibodies conjugated with horseradish peroxidase (HRP) were used at 1:10000 dilution. Immunoreactive bands were detected using ECL system. The total intensity of bands were analyzed by ImageJ. miRNA analysis. Removed fetals brain tissue in E18, and divided into hippocampus and cortex then put into TRIzol.Then, used Rat Neurological Development & Disease miRNA PCR Array (Qiagen MIRN-107Z, Germany) to analysis neuron development and neuron disease. First, miRNA reverse-transcription. Second, analyzed miRNA by qPCR array.
Statistics. All data was expressed as the mean ± SEM. Statistical significance (p < 0.05) between groups was determined using ANOVA following by the appropriate post hoc test or between two selected groups using appropriate test by GraphPad InStat software. *p < 0.05; **p < 0.01; ***p < 0.001.

Results
GMI alleviated the inflammatory responses in mother rats and their offspring for mother rats exposed to PM 2.5 during pregnancy. The white blood cell (WBC) count in pregnant rats after exposure to PM 2.5 , especially increased neutrophils; however, all types of WBCs decreased after GMI was administered in rats at different concentrations ( Fig. 1). Exposure to PM 2.5 also caused the expression of cytokines i.e., TNF-α, monocyte chemoattractant protein-1 (MCP-1), interleukin 1 beta (IL-1β), interleukin-15 (IL-15), and vascular endothelial growth factor (VEGF) significantly increased in the rats; however, GMI administration reduced the expression of these cytokines to levels comparable to control group rats (Fig. 2). These results indicated that GMI can alleviate inflammatory responses induced by exposure to PM 2.5 . Additionally, the WBC count, as well as immunoglobulin E (IgE) and histamine levels increased significantly in the offspring of female rats exposed (A,B) PM 2.5 -exposed offspring swimming trail will not center on platform. GMI shorten the latency to platform in working memory test. (C,D) in NOR and NOL test PM 2.5 -exposed offspring spend less time to explore novel object. *, and ** indicate p < 0.05, and p < 0.01 significant difference, respectively, compared with control. # , and ## show p < 0.05, and p < 0.01, respectively, compared with PM 2.5 -treated controls.
www.nature.com/scientificreports www.nature.com/scientificreports/ to PM 2.5 during pregnancy, although the offspring's WBC count declined when their mothers were administered GMI during pregnancy (Fig. 3).
GMI alleviated PM 2.5 -induced impairment of the memory capacity of offspring. The results of a behavioral tests indicated that the offspring of rats exposed to PM 2.5 exhibited a decreased performance in recognition as well as long-term, working, and spatial memory (Fig. 4). However, PM 2.5 -induced damage to the memory capacity of the offspring of mother rats administered GMI during pregnancy was reduced, leading to significant differences in working memory in comparison with rat pups whose PM 2.5 -exposed mothers were not given GMI during pregnancy.
GMI alleviated PM 2.5 -induced neurological impairments in offspring. Cerebral neural cells were studied using the Golgi method, a heavy metal-staining procedure that examines the complexity of dendritic branches to determine the robustness of synaptic cells, and the capability of the cells to process and integrate messages. The quantitative results of the Sholl analysis (Fig. 5) revealed decreases in the number of dendritic branches and terminals in CA1 and CA3 of the hippocampus as a result of exposure to PM 2.5 . Therefore, exposure to PM 2.5 decreased the number of dendritic branches and terminals. However, GMI attenuated this PM 2.5 -induced neurological damage.
GMI caused microglia to decrease in the brains of offspring. The Western blot was performed on the cortex and hippocampus of offspring to estimate the expression of antioxidant proteins (SOD-1 and catalase) and macrophages/microglia (CD 68). With exposure to PM 2.5 , the expression of SOD-1 exhibited no significantly changed, whereas that CD68 increased, particularly in the cortex (Fig. 6). Moreover, after GMI administration, microglia activation rate decreased. GMI protected miRNAs from PM 2.5 modulation, thus preventing neurological disorders. The miRNAs in the cortex and hippocampus of E18 fetuses that mother rats exposed to PM 2.5 during pregnancy were assayed through a quantitative polymerase chain reaction for neurological disorders (autism spectrum disorders, schizophrenia, anxiety disorders, Tourette syndrome, Alzheimer disease, Prion diseases, Huntington disease, Parkinson disease, and Spinocerebellar ataxia type 1). Compared with the control group, the genes in the hippocampus and cortex of offspring whose mothers were exposed to PM 2.5 were significantly modulated by air www.nature.com/scientificreports www.nature.com/scientificreports/ pollutants. These modulations could lead to neurological disorders such as schizophrenia, Prion diseases, and Alzheimer disease. The analysis results (Table 1) indicated that miRNAs in the offspring of pregnant rats had given GMI avoided modulation by PM 2.5 , making them less prone to neurological disorders.

Discussion
PM 2.5 is complex mixture whose core comprises organic carbon. Its particulates are less than 2.5 µm; it is the smallest substance that can be inhaled to cause severe damage. Once inhaled in the human body, PM 2.5 increases the risk of chronic pulmonary diseases, cardiovascular diseases, stroke, and organ impairment 16,17 . The offspring of maternal mice exposed to PM 2.5 from diesel exhaust tended to have more allergies 18 . This study revealed that after GMI administration, inflammation in mother rats throughout pregnancy eased, and WBC counts in their pups decreased (Figs 1 and 3). Moreover, according to our analysis of IgE and histamine levels in offspring GMI, especially at a dose of 0.33 μg/kg, reduced inflammatory response in rat offspring that was induced by their mothers' exposure to PM 2.5, (Fig. 3). The expression of TNF-α, MCP-1, IL-1β, IL-15, and VEGF in rats increased significantly because of their exposure to PM 2.5 during pregnancy but decreased after GMI administration (Fig. 2). Previous studies have indicated that exposure to PM 2.5 after pregnancy causes an increase in the expression of proinflammatory factors (IL-1β, IL-6, and TNF-α) 19 , which in turn leads to substantial increases in the number of neutrophils 20 . MCP-1, produced by macrophages and endothelial cells, is a monocyte chemokine that facilitates the infiltration of macrophages into adipose tissues 21,22 . Accordingly, increased monocytes in white WBCs may be a result of increased MCP-1 expression. IL-15, produced by monocytes, facilitates the generation of natural killer cells and might result in increased lymphocyte counts after exposure to PM 2.5 . Increased VEGF expression causes vascular permeability 23 , as does exposure to PM 2.5 24 ; thus, PM 2.5 passes through vessels to damage other tissues within the body. PM 2.5 leads to increases in the expression of proinflammatory factors 19 , thus intensifying oxidative stress. This study revealed that GMI administration reduced the expression of proinflammatory factors 25 , inflammatory responses, and oxidative stress in rats, and attenuated their offspring's inflammatory response.
The behavioral test results indicated that exposure to PM 2.5 impaired long-term, working, and spatial memory in rat pups (Fig. 4). This finding corresponds with epidemiological findings that PM 2.5 reduces working memory capacity 4,5 . After GMI administration, memory impairments due to PM 2.5 in offspring improved. The hippocampus is responsible for cognition, learning, and memorization. The hippocampal circuitry receives information from CA3 and sends the information downwards. The circuit terminates at CA1. CA3 is responsible for spatial Figure 6. GMI decreased microglia activation which caused by maternal PM 2.5 exposure. Immunoblot analysis determined the expression levels of microglia activation, catalase and SOD-1 in offspring cortex and hippocampus. PM 2.5 increased microglia activation and GMI significantly reduced the microglia activation in hippocampus. *, and ** indicate p < 0.05, and p < 0.01 significant difference, respectively, compared with control. # , and ## show p < 0.05, and p < 0.01, respectively, compared with PM 2.5 -treated control.
www.nature.com/scientificreports www.nature.com/scientificreports/ and working memory [26][27][28][29] . Long-term memory is the interaction between CA3 and CA1, which re-encodes and stabilizes information in the cortex [30][31][32] . The capability of neurons to process and transmit information depends on the complexity of dendritic branches. Dendritic branches in CA1 and CA3 decreased following exposure to PM 2.5 (Fig. 5). The representative images of CA1 neurons show the dendritic branches number decrease in response to PM 2.5 exposure, while GMI mitigates the branches number reduction (Fig. 5E-G). This finding agreed with the behavioral test results, in that the memory capacity of offspring declined because of exposure to PM 2.5 . Previous studies have suggested that exposure to PM 2.5 increases oxidative stress in the amniotic fluid in the bodies of mothers, thus inhibiting the growth of the fetal brain 7 . By this reasoning, exposure to PM 2.5 might have damaged the memory capacity of rat pups in this study through the same mechanism, and GMI administration might have prevented further PM 2.5 -induced increases in oxidative stress.
Microglia are immune cells in the central nervous system. As the results of the Western blot suggested (Fig. 6), high CD68 expression indicated an increased activation of microglia, and GMI administration attenuated this over activation. This increased microglial activation was probably due to elevated levels of inflammatory factors in peripheral tissues, which activate cerebral endothelial cells, affecting immune cells in the central nervous system 33 . Intense inflammation in offspring may arise from the inflammatory responses of mothers during pregnancy; moreover, the immature blood-brain barrier in the fetus leads to inflammation in its brain, thus inhibiting its development. Inflammation during pregnancy can lead to hippocampal abnormalities in the fetus, such as loss of neurons, astrocyte multiplication, and altered expression of neurotransmitter receptors 34 . Astrocyte multiplication causes microglia to increase, thus inducing neurological damage 35 ; this accounts for neurological damage and memory impairment in rat offspring whose mothers are exposed to PM 2.5 during pregnancy.
An miRNA expression analysis indicated that when mothers were administered GMI during pregnancy, miR-NAs in offspring with schizophrenia, Prion diseases, and Alzheimer disease were not modulated by PM 2.5 ; only rno-miR-9a-3p, which is related to schizophrenia, underwent this modulation (Table 1).
In summary, exposure to PM 2.5 during pregnancy not only induces inflammatory responses in mother rats but also leads to memory impairment and cognitive decline in offspring as well as the overactivation of their microglia. GMI administration can alleviate the inflammatory responses, impaired memory capacity, and neurological damage induced by PM 2.5 . Therefore, despite exposure to PM 2.5 during pregnancy causing long-term damage to the fetus, GMI administration can safeguard mothers and their offspring from PM 2.5 -induced damage.