Effect of Serum Bisphenol A and Low T3 on Short-Term Prognosis of Young Patients with Intracerebral Hemorrhage

Background Bisphenol A (BPA) is an environmental chemical substance, and the widespread use of plastic products, its hazards can be said to be ubiquitous. We aimed to evaluate the relationship between serum bisphenol A (BPA), biomarkers and recent mortality in pats with young interacerebral hemorrhage (YICH) and different thyroid functions,and the cumulative consumption of plastic products. Using a multi-center longitudinal prospective study, the research team recruited 600 YICH patients and divided them into two subgroups according to their thyroid function, of which 320 (53.3%) were in the low T3.Sub-clinical hypothyroidism(SCH) was 280 (46.6%) and control group (n = 600).All patients received serum BPA, and biomarker blood draws. together to construct an inammatory response program, is related to the risk of short-term death. There is a signicant difference in recent mortality compared to LT3 vs SCH. insights. BPA is an industrial chemical that exists widely in the environment. Humans are exposed to BPA through food, milk, and skin contact [6, 7 ] . High levels of BPA exposure are related to abnormal regulation of thyroid ,autoimmune functions, obesity and metabolism [8, 9 ] . However, at current exposure levels in developing world the link between ICH and BPA remain unproven.This study focuses on clarifying the relationship between the cumulative quantication of BPA exposure levels and the risk of ICH, and assessing the correlation between different thyroid function,ambulatory blood pressure (ABP),miRNA,biomarkers and recent mortality. marital hypertension The of interaction analysis showed that there is a multiplicative and additive interaction between whether serum BPA is related to nocturnal hypertension (U = 4.19,


Abstract
Background Bisphenol A (BPA) is an environmental chemical substance, and the widespread use of plastic products, its hazards can be said to be ubiquitous. We aimed to evaluate the relationship between serum bisphenol A (BPA), biomarkers and recent mortality in pats with young interacerebral hemorrhage (YICH) and different thyroid functions,and the cumulative consumption of plastic products.

Methods
Using a multi-center longitudinal prospective study, the research team recruited 600 YICH patients and divided them into two subgroups according to their thyroid function, of which 320 (53.3%) were in the low T3.Sub-clinical hypothyroidism(SCH) was 280 (46.6%) and control group (n = 600).All patients received serum BPA, and biomarker blood draws.

Conclusion
Thus, Long-term exposure to BPA levels may be related to the occurrence of YICH, High BPA exposure strongly predicts the risk of ICH in young patients. Activation of NF-κB signaling pathway, secondary thyroid function and ABP changes, together to construct an in ammatory response program, is related to the risk of short-term death. There is a signi cant difference in recent mortality compared to LT3 vs SCH.

Trial registration
The trial is registered at clinical .gov (www.medresman.Chi CTR1900023626) Introduction ICH accounts for 10%-15% of stroke cases, and the assessment of ICH risk factors is clinically challenging [1,2] . Common mutations in gene locus, vascular risk factors, peripheral gene toxicity and dynamic changes of immune cells are associated with stroke risk [3,4] . For this reason,exploring the unique eld of interaction of self-protective neuroimmune in ammation will likely bring strategic positioning to the prognosis or treatment of ICH 5 . This study focuses on the changes in the levels of BPA gene toxicity and neurotoxicity and in ammatory markers, in order to further evaluate the relationship between the targeted proteases in ICH and the biological trigger BPA, which will help patients with ICH Neurotoxic molecular research provides in-depth new insights.
BPA is an industrial chemical that exists widely in the environment. Humans are exposed to BPA through food, milk, and skin contact [6,7 ] . High levels of BPA exposure are related to abnormal regulation of thyroid ,autoimmune functions, obesity and metabolism [8,9 ] . However, at current exposure levels in developing world the link between ICH and BPA remain unproven.This study focuses on clarifying the relationship between the cumulative quanti cation of BPA exposure levels and the risk of ICH, and assessing the correlation between different thyroid function,ambulatory blood pressure (ABP),miRNA,biomarkers and recent mortality.

Patient
Analyze the clinical data of 600 YICH patients admitted to the Department of Intensive Care and Neurology,Changzhi People's Hospital. Changzhi Medical College Peace Hospital and Changzhi Second People's Hospital from January 2011 to January 2014. All selected cases are used Systematic sampling, multi-center and randomized controlled studies. According to the level of thyroid function, they were divided into two subgroups, including 320 (53.3%) in the LT3 and 280 (46.6%) in the SCH. Meets the criteria of the American Stroke Association,Clinical Neurology Committee 10 . Exclusion criteria: (1) Liver and kidney damage, visceral malignant tumors, history of diabetes, family history of hypertension and previous use of aspirin. (2) Patients with cerebrovascular malformations and cerebrovascular tumors found in computed tomography must be excluded through cerebrovascular angiography.
The normal control group is selected strictly according to the screening criteria: a history of long-term exposure to plastic products are not eligible, healthy individuals (n = 600) matching age and gender .

Follow-up data
The methods include household entry, community visits, telephone calls, and reporting. Investigation of long-term exposure to plastic products: understand the use of plastic bottles from the time of infancy, children, adolescents, and adults who have habitually contacted or used plastic bags, especially the fried dough sticks were taken out of the oil pan and put into the plastic bag for breakfast. The establishment of product quantitative. Additionally, collect routine life data, including patient medication history, smoking and drinking history, body mass index (BMI): BMI = weight (kg) /height 2 (m 2 ). Smoking is de ned as continuous cumulative smoking for 6 months or more; drinking is de ned as drinking at least one glass of wine in the past 30 days .

Serum analysis
Serum samples were collected on an empty stomach in the morning on the 2, 15 and 30 days after admission.The enzyme-linked immunosorbent assay measures BPA, thrombin,and MMP-9 from Shenzhen Galvanomi Biotechnology Co, Ltd. Provided; Electrochemiluminescence immunoassay measurement of total testosterone and free testosterone are provided by Bayer;triiodothyroxine (T3) and free triiodothyroxine(FT3), thyroxine (T4), free thyroxine (FT4) and Thyroid Stimulating Hormone (TSH) provided by Shanghai Science Co, Ltd.
TLR4-NF-κB signaling pathway related protein expression : Collect peripheral venous blood, separate mononuclear cells, use Western Blot method to detect, Toll-like receptor 4 (TLR-4), Myeloid differentiation factor 88 (My88),(TRAF6)tumor necrosis factor-associated factor6 ,nuclear transcription factor κBp65 (NF-κBp65).The protein was extracted from mononuclear cells with RIPA lyase buffer containing protease inhibitors, and the color was developed using a chemiluminescence kit. Perform quantitative analysis on the image and calculate the relative expression of each protein.
Expression of miRNA338-3p and miRNA144/451 in peripheral blood lymphocytes Take 2ml of fasting venous blood was drawn from the survey subjects, and total RNA was extracted by the Trizol method. Real-time uorescence quantitative polymerase chain reaction (PCR) method to detect the expression level of miRNA: using total RNA as a template, using random primers for reverse transcription ampli cation synthesis,obtaining miR-338-3p,miR144/451 internal reference Gene glyceraldehy-de 3-phosphate dehydrogenase (GAPDH) RNA d cDNA template; speci c PCR primers were used to detect the expression of miRNA and GAPDH genes, and the 2-△△ct method was used to analyze the expression of indicators.

Statistical analysis
General demographic data is the mean ± SD of continuous variables and the percentage of categorical variables. The qualitative data comparison adopts oneway analysis of variance and chi-square test, and the main statistical indicators are tested for normality. First, the Mann-Whitney µ test is used to assess the average difference between the two groups. The comparison of the means of the two large samples of the group design uses the µ test. The comparison of the three sets of data uses F test. The changes of serum BPA concentration, cytokines, protease and 24hBP were analyzed by linear correlation analysis, regression analysis was used to determine the correlation between BPA concentration and thyroid function ; through multiple linear regression analysis, the in uence of various factors on the percentage of nSBP and nDBP was analyzed. Use receiver operating characteristic curve (receiver operating characteristic curve, ROC curve) to de ne the boundary value of miRNA high and low expression level; use cross-sectional analysis combined with Logistic regression model to analyze the combination of serum BPA, ABP and miRNA expression levels, cross-effects, and calculate relative Risk ratio attribution ratio and interaction index. Using SPSS v20.0 software (SPSS Inc, USA) for statistical analysis, the difference was statistically signi cant, P<0.05.

Results
There were 240 males and 80 females in the LT3 group (75% vs 25%); 80 males and 200 females in the SCH group (28,57% vs 71.42%); the average age was 25±7.2 years, and the control group was 25.3±6.6 years old. Compared with the control group, 24hSBP, 24hDBP, insulin resistance-index, blood lipids, etc.
increased in all YICH patients, especially nDBP increased signi cantly, P <0.05. Additionally, calculating the cumulative amount of plastic bags for 10 years, each bag is 1.6 grams, 3000 bags, the total is about 4800g. For every log unit increase in BPA, it is determined that the Z score of the plastic bag increases by 0.204 standard deviation units (P<0.003). (Table 1) ICH patients and the control were divided into two gender subgroups. The BPA level of ICH men increased signi cantly compared with women and the control, and there was also a difference between women and the control p <0.01 . (Figure 1 ) LT3-male nDBPcv compared with female and control, SCH male, female compared with control group, P<0.01. (Figure 2) Compared with the female LT3 and the control, the FT3 of the male LT3 was signi cantly reduced, and the female SCH group TSH was signi cantly increased compared with the male and the control, P<0.05 . (Figure 3) There were 600 cases of ICH, and the recent total mortality rate was 74 cases (12.3%). There were 320 cases of LT3 and 50 deaths (15%); 280 cases of SCH and 24 deaths (8.57%). The recent mortality rates of the two groups were compared in 5, 15, and 30 days.15 (4.7) vs 7(2.5),20(6.25) vs 10 (3.75),15(4.68)vs 7(2.5) P<0.01. (Figure 4) On day 2, serum miR-338-3p signi cantly increased compared with the control, while miR-445 signi cantly decreased. P<0.01. (Figure 5) On day 15 serum miR-338-3p signi cantly lowed compared with the control,while miR-451 signi cantly increased.P<0.01. (Figure 6) Serum miR-338-3p decreased more signi cantly on day 30 compared with that on day 15 and the control, while miR-451 increased signi cantly. P<0.01. (Figure 7) Compared with the control, the levels of serum BPA, various proteases, in ammatory cytokines and miR-338-3p on day 2 of LT3 and SCH increased, miR-455 decreased, and blood pressure parameters increased, P<0.05. The ratio of free testosterone in male patients to the control group was signi cantly lower P <0.01. In addition, there was no signi cant difference in serum rT3 and miR-144 levels between the two groups, P≥0.05, omitted. (Table 2) Compared with the control, the serum BPA and protease in the LT3 and SCH groups at 15 and 30 days tended to decrease; on the contrary, the level of miR-338-3p decreased, the level of miR-455 increased, and the blood pressure parameters increased. In particular, nDBPCV was more Obviously, P <0.01. (Table 3) Serum BPA is positively correlated with biomarker, 24hDBPCV, and was negatively correlated LT3 and niR-451. (Table 4) The increase of nSBP and nDBP were used as dependent variables, and age, gender, BMI and BPA were used as independent variables. The results of multiple linear regression showed that cumulative BPA exposure was an independent factor affecting of nSBP and nDBP. (Table 5) The relationship between BPA, thrombin and miRNA expression risk factors: draw a BPA ROS curve, the area under the curve is 0.744 (95% CI 0.624-0.894), the maximum Uden index is 0.426, the sensitivity is 75.4%, and the speci city is 67.4%, corresponding to a cut-off value of 1.567. Adjusting for confounding factors such as gender, age, and marital status, the risk of BPA in ICH patients is 5.12 times that of the general population. (Table 6)   Increase BPA levels (none = 0, yes = 1) and miR-451 (none = 0, yes = 1), nDBPCV (none = 0, yes = 1), binding and interaction analysis. The results of the crosshealth analysis showed that after adjusting for factors such as gender, age, and marital status. Nocturnal hypertension and the low expression of miR144/451 and the presence of elevated serum BPA are signi cantly different from the BPA levels of patients with non-nocturnal hypertension, and there is a risk of ICH.
The results of interaction analysis showed that there is a multiplicative and additive interaction between whether serum BPA is related to nocturnal hypertension (U = 4.19, P <0.05) and whether miR-451 is under-expressed (U = 6.16, P <0.05)(

Discussion
Bisphenol A (BPA) is an endocrine disruptor, which not only interferes with thyroid function, but also affects sexual function. Studies have reported [12][13][14] that in stress diseases such as heart and brain, female patients are mostly at risk of SCH This study found that the serum BPA in the male group of YICH patients was signi cantly increased compared with the female group, and the free testosterone (FT) level in men was signi cantly reduced. It also shows that women are mostly SCH, while men are mostly LT3 syndrome. It is speculated that there is a certain inherent correlation between the toxic effects of BPA exposure on the thyroid and sexual function, involving a chain of chain events.
Ambulatory blood pressure is related to target organ damage, and the circadian rhythm of blood pressure has an important in uence on the pathophysiology of diseases. Studies have reported that [15][16][17] BPA exposure from canned food can increase blood pressure, and blood pressure variability is involved in the occurrence and development of target organ damage [18] . Although BPA exposure does not directly affect plasma lipid levels, it can enhance the expression of CD36 in macrophages, which is related to lipid accumulation [19] . There are changes in oxidized protein and lipid levels in atherosclerosis [20] . The baseline data of this study showed that the blood lipid level of YICH patients was signi cantly higher than that of the control, and the night diastolic blood pressure and blood pressure scores were signi cantly increased. In addition, multiple linear regression analysis showed that BPA is an independent risk factor that affects the percentage increase of nSBP and nDBP.
The biological trigger BPA is biochemically toxic to multiple organ functions. Exposure is related to oxidative stress, insulin resistance, and changes in microRNA (miR) expression pro les [21,22] , de ning the potential mechanism of toxic miR. miR-144/451 may be bene cial under stress conditions and has a certain protective effect on vascular endothelial function. Oxidative stress is a series of reactive oxidation, forming a series of network links from biological toxicity to cytotoxicity and lipotoxicity.
In ammation and apoptosis are related to the prognosis and survival of patients, and the in ammatory environment can cause neuronal apoptosis [23] . BPA exposure can induce vascular toxicity and risk events, which can gradually affect cerebrovascular pathology and molecular phenotypic biochemical factors.
Nuclear factor-κB transcription factor is a multifunctional transcription factor. The NF-κBp65 in ammatory pathway induces oxidative stress and apoptosis, and improves cytological functions [24] . In ammation, oxidative stress and other factors can cause vascular dysfunction and structural damage [25] . Therefore, targeting TLR4 in ICH patients can attenuate the in ammatory response, thereby attenuating apoptosis and improving prognosis [26] .The study of this group showed that serum in ammatory cytokines and TLR4-NF-κB signaling pathway series factors were signi cantly increased, and the initial thrombin concentration was associated with a higher risk after stroke, indicating that the expression of ncRNA has time-point changes. It is speculated that this phenomenon is related to the treatment and the protective factors of the body's autoimmunity. This study also found that the recent mortality rate of the LT3 group was signi cantly increased compared with that of the SCH group, indicating the resonance of the body, which needs to be con rmed by further studies.
In addition, correlation analysis showed that BPA was positively correlated with miR-338-3P and a variety of proteases, and negatively correlated with miR-451.
The main limitation of this study is the lack of analysis and research on the relationship between the drug and BPA biotoxicity and biomarkers. In-depth analysis of the anti-in ammatory, network homeostasis and neuroprotective effects of connexin 43 after ICH injury is required. Clarify the innervation or position of β3 adrenergic nerves, and target the adrenergic system to effectively inhibit the neuroin ammatory mechanism after ICH Conclusion High BPA exposure strongly predicts the risk of YICH patients. Activation of NF-κB signaling pathway, secondary thyroid function and ABP changes, together to construct an in ammatory response program, is related to the risk of short-term death.

Declarations
Availability of data and materials The de-identi ed dataset generated and analyzed in this study is electronically stored and protected by speci c password. The dataset is available from the corresponding author on reasonable request.  Comparison of mortality between the two groups Figure 5 Comparison of miR-338-3p ,miR-451 levels in the two groups (2 days. a,b, c,d Figure 6 Comparison of miR-338-3p ,miR-451 levels in the two groups (15days. a,b, c,d Comparison of miR-338-3p ,miR-451 levels in the two groups (30 days. a,b, c,d

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. BPA2.doc