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Abstract. – OBJECTIVE: To investigate the effect of tauroursodeoxycholic acid (TUDCA) on neurological impairment induced by acute cere-bral infarction (ACI) and its relevant mechanism of action.

PATIENTS AND METHODS: A total of 60 male Sprague-Dawley (SD) rats were randomly di-vided into Sham group (n = 20), ACI group (n = 20), and TUDCA group (n = 20). The rat model of ACI in middle cerebral artery was established. TUDCA was intravenously injected into rats in the TUDCA group, while an equal amount of so-dium bicarbonate solution was intravenously injected into the other two groups. The blood was drawn after modeling to detect the content of serum glutamate (Glu), triglyceride (TG), to-tal cholesterol (TC), and low-density lipopro-tein cholesterol (LDL-C). The degree of cere-bral infarction in each experimental group was observed under an optical microscope, and the infarct area was measured and compared. The content of serum tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and high-sensitivi-ty C-reactive protein (hs-CRP) was detected via enzyme-linked immunosorbent assay (ELISA); mRNA and protein expressions of them were detected using reverse transcription-poly-merase chain reaction (RT-PCR) and Western blotting, respectively, followed by statistical analysis. Moreover, the expression levels of se-rum malondialdehyde (MDA), oxidized-LDL (ox-LDL), superoxide dismutase (SOD), and glu-tathione peroxidase (GPX) were detected, fol-lowed by statistical analysis. The protein ex-pressions of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), very low-density lipoprotein receptor (VLDLR), nuclear factor-κB (NF-κB), B-cell lymphoma 2-associated X protein (Bax), and caspase-3 were detected via Western blot-ting, and the gray value was determined, fol-lowed by statistical analysis.

RESULTS: TUDCA could improve the symp-toms of neurological impairment in ACI patients, decrease the National Institute of Health Stroke Scale (NIHSS) score but increase the activity of daily living (ADL) score of patients, and signifi-cantly reduce the content of serum TG, TC, and LDL-C, showing statistically significant differ-ences (p < 0.05). TUDCA significantly decreased the serum Glu content in ACI rats, reduced the cerebral infarction area and lowered the se-rum TG, TC, and LDL-C content, displaying sta-tistically significant differences (p < 0 .05). Be-sides, TUDCA inhibited mRNA and protein ex-pressions of TNF-α, IL-8, and hs-CRP, and allevi-ated the inflammatory response. TUDCA inhibit-ed MDA and ox-LDL expressions, but increased SOD and GPX expressions, and relieved oxi-dative stress injury. In addition, TUDCA could negatively regulate Nrf2 signaling pathway, and down-regulated VLDLR and NF-κB protein ex-pressions and expressions of apoptotic pro-teins (Bax and caspase-3).

CONCLUSIONS: TUDCA can alleviate the ACI-induced neurological impairment in rats through mitigating lipid peroxidation and in-flammatory response and reducing apoptosis, whose relevant mechanism may be that TUD-CA negatively regulates Nrf2 signaling pathway.

Key Words:TUDCA, ACI, Nrf2, Oxidative stress.

Introduction

Acute cerebral infarction (ACI) is one of the most common diseases in neurology1. Its inci-dence, disability and mortality rates are increased year by year with the improvement of people’s

European Review for Medical and Pharmacological Sciences 2019; 23: 343-351

K.-Y. BIAN1, H.-F. JIN1, W. SUN2, Y.-J. SUN3

1Department of Neurology, Wujin Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Changzhou, China2Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China3Department of Pathology, Affiliated Jiangyin Hospital of Southeast University Medical College, Jiangyin, China

KeyuBian and Huafeng Jin contributed equally to this work

Corresponding Author: Yuejun Sun, MM; e-mail: vduy51@163.com

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living standard and changes in dietary structure. Atherosclerosis of the vascular wall leads to the lumen obstruction, thus causing hypoxia and necrosis of brain tissues and resulting in a series of neurological impairments, which is one of the major causes of ACI2. It is known that the infiltra-tion of inflammatory mediators plays key roles in the formation of atherosclerosis, and the resulting inflammatory cascade increases the degree of ACI3. There are also reports that the formation of atherosclerosis is accelerated by lipid deposi-tion induced by high-fat diet or lipid metabolism disorders4. For example, low-density lipoprotein cholesterol (LDL-C) can increase the artery in-tima-media thickness, accelerate lumen steno-sis, and increase the risk of ACI5. A significant amount of high-sensitivity C-reactive proteins (hs-CRPs) deposit in atherosclerotic plaques, and will be much released in the occurrence of ACI, damaging the vascular endothelium6. Moreover, studies have also proved that hs-CRP can pro-mote the release of inflammatory factors7, such as tumor necrosis factor-α (TNF-α) and interleu-kin-8 (IL-8), thereby exacerbating the inflamma-tory response. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway has been con-firmed to be one of the most important signaling pathways regulating inflammatory response and lipid metabolism disorders8. In addition, it has been identified that very low-density lipoprotein receptor (VLDLR) accumulates abnormally in liver cells9, thus causing alcoholic fatty liver. The expression of VLDLR is regulated by Nrf2 signaling pathway, which can be activated by oxidative stress8. Scholars11-12 have also revealed that the ACI-induced damage is associated with the excessive apoptosis of brain tissues and cells caused by ischemia and hypoxia. It was acciden-tally found in clinical work that the prognosis of ACI patients taking tauroursodeoxycholic acid (TUDCA) is superior to that of patients without TUDCA. It has been confirmed11 that TUDCA can alleviate the neurological impairment in rats with acute stroke, but its relevant mechanism remains unclear.

TUDCA possesses anti-inflammatory and an-ti-oxidative effects and definite efficacy in the treatment of reflux gastritis, which has been proved to be able to inhibit the bile acid-induced liver cell apoptosis via Bax and caspase-3 signal-ing pathways13. Therefore, it is speculated that the effect of TUDCA on ACI patients is possibly related to its anti-inflammatory, anti-oxidative and anti-apoptotic effects and its influence on

the Nrf2 signaling pathway. Hence, this animal experiment was performed to investigate the ef-fect of TUDCA on ACI rats and its underlying mechanism.

Patients and Methods

Patients and Treatments A total of 30 ACI patients, including 21 males

and 9 females aged (56 ± 5.3) years old, in our hospital from January to April 2018 were selected and randomly divided into Group A and Group B. Inclusion criteria: (1) patients meeting diagnostic criteria of ACI and diagnosed via brain computed tomography (CT) or magnetic resonance imag-ing (MRI), (2) patients with first-onset disease, (3) patients with high compliance who could complete all examination items in this work, (4) patients not complicated with other diseases, and (5) the duration from onset to admission ≤ 48 h. Exclusion criteria: (1) patients accompanied by other heart diseases or hepatic-renal dysfunction, (2) patients with a previous history of stroke, (3) patients with coagulation disorders or bleeding tendency, or (4) patients with allergic reaction or intolerance to drugs used in this study. After admission, the blood was drawn from patients to detect the content of serum triglyceride (TG), total cholesterol (TC), and LDL-C; the National Institute of Health Stroke Scale (NIHSS) score and the activity of daily living (ADL) score were given. After that, Group A was treated with basic treatment, while Group B received basic treat-ment and oral administration of TUDCA (100 mg/time and 3 times/day). The above indexes were detected again after 1 week, followed by the statistical analysis. This study was approved by the Ethics Committee of Affiliated Jiangyin Hos-pital of Southeast University Medical College. Signed written informed consents were obtained from all participants before the study.

Animals and TreatmentsA total of 60 male Sprague-Dawley (SD) rats

weighing 180-200 g purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were randomly divided into Sh-am group (n=20), ACI group (n=20), and TUDCA group (n=20). After fasting for 8 h, rats in each experimental group were anesthetized with 10% chloral hydrate (0.3 g/kg). Under a supine posi-tion, the carotid artery of rats in Sham group was isolated. In the other two experimental groups,

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the carotid artery of rats was also isolated, the distal external carotid artery was ligated, the external carotid artery stump was cut near the bi-furcation, and the silicon wire used for the thread occlusion was inserted into the internal carotid artery and extended for 18-20 cm until the middle cerebral artery to block the blood supply in it. 2 h after operation, ischemia-reperfusion was per-formed. After recovery from anesthesia, whether there was contralateral limb paralysis of rats was determined to judge the success of modeling, and rats who were modeled unsuccessfully or dead were excluded from the study. TUDCA solution at a concentration of 400 mg/mL was prepared using 0.15 mmol/L sodium bicarbonate (pH= 7.4). 1 h after modeling, rats in TUDCA group were intravenously injected with TUDCA (400 mg/kg), while those in the remaining experimental groups were injected with an equal amount of sodium bicarbonate solution. 3 d after the oper-ation, rats in each experimental group were exe-cuted via cervical dislocation, and the blood was taken, centrifuged at 3000 rpm for 10 min and stored in a refrigerator at -80°C for standby ap-plication. The skull was opened to take the brain tissues, some of which was fixed in 4% formalin, and some were cut into small pieces and stored at -80°C for standby application. This research was approved by the Animal Ethics Committee of Af-filiated Jiangyin Hospital of Southeast University Medical College Animal Center.

Histopathological Observation of Serum Glutamate (Glu) and TG, TC, and LDL-C

The above serum was taken to detect the con-tent of serum Glu and TG, TC, and LDL-C using a full-automatic biochemical analyzer (KeyGEN, Nanjing, China). Brain tissues fixed in 4% for-malin were embedded in paraffin and sliced into sections. After hematoxylin and eosin (H&E) staining, the infarct area and cellular edema in the same field of view of the rat’s brain tissues in each experimental group were observed under an optical microscope (×400), followed by statistical analysis.

Detection of the Content of hs-CRP, TNF-α, and IL-8 and Their mRNA andProtein Expressions

After 1 mL serum already prepared in each ex-perimental group was taken, the content of serum inflammatory factors was measured using the enzyme-linked immunosorbent assay (ELISA) kit (purchased from Shanghai Bohu Biological

Co., Ltd. Shanghai, China). An equal number of brain tissues were taken and ground into tissue homogenate, and total RNA was extracted using the RNA kit. Relevant primer sequences were searched in PubMed database (Table I), and the extracted RNA was reversely transcribed into complementary Deoxyribose Nucleic Acid (cD-NA), followed by amplification via reverse tran-scriptase-polymerase chain reaction (RT-PCR) to detect its expression. Finally, the mRNA expres-sion in each experimental group was calculated using the 2-ΔΔCT method, and the protein expres-sion was detected via Western blotting14. Glycer-aldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control in the experiment. Experimental antibodies were purchased from Abcam (Abcam, Cambridge, UK).

Detection of Serum Oxidative Products (MDA and ox-LDL) and Antioxidants (SOD and GPX)

An equal amount of serum was taken from each experimental group. The content of malond-ialdehyde (MDA) was determined using the thio-barbituric acid (TBA) colorimetric method shown in the literature, while the activity of superoxide dismutase (SOD) was detected using the nitrite method15. The content of oxidized-LDL (ox-LDL) and glutathione peroxidase (GPX) was measured using the full-automatic biochemical analyzer (KeyGEN, Nanjing, China) according to instruc-tions.

Detection of Brain Tissue Protein Via Western Blotting and Determination of Its Gray Value

After the cryopreserved brain tissues of rats were thawed and ground into tissue homoge-nate, the expression levels of related proteins [Nrf2, VLDLR, nuclear factor-κB (NF-κB), Bax, and caspase-3] were detected via Western blotting14. GAPDH was used as the internal reference, and experimental antibodies were

Table I. Primer sequences.

Gene Sequence

IL-8 F: 5′-CATTAATATTTAACGATGT GGATGC-3′ R: 3′-TAACACGTCAAATTTCTACCATCCG -5′TNF-α F: 5′-CATCCGTTCTCTACCCAGCC-3′ R: 3′-AATTCTGAGCCCGGAGTTGG-5′hs-CRP F: 5′-CCTCTGGATACAGCTGCGAC-3′ R: 3′-GTAGATGCCGGGTGGTTCAA-5′

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purchased from Abcam (Abcam, Cambridge, UK). The gray value of each protein was deter-mined using Quantity One software, followed by statistical analysis.

Statistical AnalysisData obtained in the experiments were ana-

lyzed using Statistical Product and Service Solu-tions (SPSS) 19.0 software (IBM, Armonk, NY, USA), and presented as mean ± standard devi-ation. The t-test was applied for the data com-parison between two groups. p<0.05 suggested that the difference was statistically significant. GraphPad Prism 5.0 (La Jolla, CA, USA) was used for plotting.

Results

TUDCA Could Significantly Improve the Symptoms of Neurological Impairment and Reduce the Blood Lipid Level in ACI Patients

The NIHSS score and ADL score of ACI patients were compared between Group A and Group B before admission and after application of TUDCA (Figure 1A), and differences were statistically significant (p < 0.05), indicating that the patient’s neurological impairment and quality of life are significantly improved after application of TUDCA. Besides, the levels of serum lipid, TG, TC, and LDL-C were all lower than those before drug administration (Figure 1B), and there were statistically significant dif-ferences (p < 0.05). The following experimental animal study was performed to study its under-lying mechanism.

TUDCA Could Significantly Alleviate the Neurological Impairment, Reduce the Blood Lipid Level and Mitigate Pathological Changes in Brain Tissuesof ACI Rats

Glu is one of the key markers of neurological impairment16, which will be increased by hy-poxia to produce neurotoxicity, causing nerve damage. Results of this investigation revealed that compared with that in ACI group (Figure 2B), the serum Glu level in experimental rats in TUDCA group was decreased, and the dif-ference was statistically significant (p < 0.05). The degree of cerebral infarction affects the prognosis of ACI patients directly3. Therefore, the degree of cerebral infarction in each exper-imental group was observed under the optical microscope (Figure 2D), and the cerebral infarc-tion areas in several fields of view were mea-sured (Figure 2), followed by statistical analysis (p < 0.05). Results showed that TUDCA could remarkably improve the degree of cerebral in-farction in ACI rats. The above results indicate that TUDCA can alleviate the neurological im-pairment in ACI rats. The comparisons of serum TG, TC, and LDL-C (Figure 2A) content in each experimental group manifested that they were significantly decreased in TUDCA group compared with those in ACI group, suggesting that TUDCA can improve the blood lipid level in ACI rats.

TUDCA Inhibited mRNA and Protein Expressions of TNF-α, IL-8, and hs-CRP and Alleviated Inflammatory Response

Hs-CRP is an important mediator in the oc-currence of ACI7, which can promote the release of such inflammatory factors as TNF-α and IL-

Figure 1. Comparisons of NIHSS/ADL scores and serum TG/TC and LDL-C content in patients between Group A and Group B at admission show no statistically significant differences (*p > 0.05). The above indexes are compared after treatment for 1 week and display statistically significant differences (*p < 0.05).

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8 and aggravate the neurological impairment3. The experimental results demonstrated that the contents of serum hs-CRP and inflammatory factors (TNF-α and IL-8) were significantly decreased compared with those in ACI rats (Figure 3A), and differences were statistically significant. Furthermore, the mRNA and protein expressions were detected via RT-PCR (Fig-

ure 3B) and Western blotting (Figure 3C, 3D), respectively. It was found that TUDCA could reduce hs-CRP, transcription, and translation of TNF-α and IL-8 in ACI rats, and there were statistically significant differences (p < 0.05). The above results indicate that TUDCA can al-leviate the inflammatory cascade and reduce the ACI-induced damage.

Figure 2. The blood is taken from each experimental rat to detect the content of TG/TC and LDL-C (a) and serum Glu (b), followed by statistical analyses, displaying statistically significant differences (*p < 0.05); (c) the cerebral infarction areas in several fields of view in 3 different groups were measured and results showed statistical difference (p < 0.05).

Figure 3. (A) Comparisons of serum TNF-α/IL-8/hs-CRP levels in experimental rats display statistically significant differences (*p < 0.05). (B) Expression levels of inflammatory factors (TNF-α/IL-8/hs-CRP) in rats in each experimental group are detected via RT-PCR, followed by statistical analysis, and there are statistically significant differences (*p < 0.05). (C) Protein expressions of inflammatory factors in rats in each experimental group are detected via Western blotting, their gray values (D) are measured and analyzed statistically, and there are statistically significant differences (*p < 0.05).

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TUDCA Could Inhibit the Expressions of MDA and ox-LDL, Increase the Expressions of SOD and GPX, and Alleviate the ACI-Induced Oxidative Stress Injury

Oxidative stress plays an important role in the occurrence and development of ACI17. Ox-ygen radicals interact with lipids to produce oxidative stress products (MDA and ox-LDL), thus aggravating the neurological impairment. In this experiment, the content of serum MDA and ox-LDL was decreased compared with those in ACI rats (Figure 4A), and differences were statistically significant (p < 0.05). However, the expression levels of antioxidants (SOD and GPX) were increased, and there were statis-tically significant differences (p < 0.05). The above results suggest that TUDCA can alleviate the oxidative stress response, thus reducing the ACI-induced damage.

TUDCA Reduced the Expressions of VLDLR and NF-κB Proteins and Down-Regulated the Expressions of Apoptotic Proteins (Bax and caspase-3) Through Negative Regulation of Nrf2 Signaling Pathway

Experimental results (Figure 5) manifested that Nrf2, VLDLR, and NF-κB protein expres-sions were decreased compared with those in ACI rats, and the differences in the gray value were statistically significant, suggesting that TUD-CA may alleviate the inflammatory response through the negative regulation of Nrf2-NF-κB inflammatory pathway, and reduce the lipid peroxidation through the negative regulation of Nrf2-VLDLR pathway. Moreover, expressions of apoptotic proteins (Bax and caspase-3) were significantly decreased in TUDCA group, in-

dicating that TUDCA can inhibit ACI-induced apoptosis. The above results suggest that the negative regulation of Nrf2 signaling pathway may be one of the mechanisms of TUDCA in alleviating ACI damage.

Discussion

In clinical work, the NIHSS score of ACI patients was decreased, but the ADL score was increased after administration of TUDCA for 1 week (100 mg/time, 3 times/day) (Figure 1A). Those results indicated that TUDCA can sig-nificantly improve the symptoms of neurological impairment and increase the quality of life of ACI patients, reducing the serum lipid level and the recurrence risk of ACI in patients (Figure 1B). This experimental study was conducted to study the role and relevant mechanism of TUDCA.

Glutamate is a kind of excitatory amino acid, which will be increased by hypoxia to produce neurotoxicity, thus leading to a variety of neu-rological symptoms18. In this experiment, the Glu content in TUDCA group was significantly reduced compared with that in ACI group (Fig-ure 2B), suggesting that TUDCA can reduce the Glu accumulation in ACI rats, thus alleviating the nerve injury. It is generally believed that atherosclerosis is the main cause of ACI2,19, and the formation of arteriosclerosis is closely relat-ed to lipid metabolism disorders4. The content of TG, TC, and VLDLR in rats in the TUDCA group was significantly reduced, suggesting that TUDCA can regulate lipid metabolism, lower the blood lipid level, and reduce the risk of arte-riosclerosis. The observation of the pathological changes in brain tissues of rats in each experi-mental group under the light microscope showed

Figure 4. Comparisons of (A) content of oxidative stress products (MDA/ox-LDL) and (B) levels of SOD and GPX in each experimental group display statistically significant differences (*p < 0.05).

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that TUDCA could reduce the degree of cerebral infarction in ACI rats. There have been reported20 that the ACI-induced neurological impairment is closely related to the excessive apoptosis of brain cells. In conclusion, TUDCA has definite effects on alleviating neurological impairment of ACI, reducing cerebral infarction and lowering blood lipids. Furthermore, the mechanism research was performed.

It is generally thought that the vascular endo-thelial injury can activate inflammatory chemo-kines2, thus inducing the inflammatory cascade and promoting the formation of atherosclerosis. IL-8 has been proved to play an important role in the occurrence and development of ACI, which not only can lead to vascular wall injury, but also has the effects of coagulation and vascular contraction21. TNF-α can activate and amplify the inflammatory response and result in cytotoxic cerebral edema, thereby aggravating brain tis-sue damage22. Studies have confirmed that after TNF-α is injected into the lateral cerebral ventri-cle of the rat model of local ischemia, both brain tissue necrosis and edema areas are increased23. Moreover, according to the literature report6, hs-CRP is a kind of acute-phase reactive protein, which can be much released in ACI. It has been reported24 that the increased concentration of hs-CRP can promote the release of a large number of inflammatory factors, such as IL-8 and TNF-α, and activate the immune complement, accelerat-ing the occurrence of atherosclerosis and raising the risk of ACI. In this work, it was found that the content of serum hs-CRP, IL-8, and TNF-α

in rats in TUDCA group were decreased (Figure 3A). Results of RT-PCR and Western blotting revealed that TUDCA could negatively regulate expressions of hs-CRP, IL-8, and TNF-α. In ad-dition, oxidative stress is another cause of athero-sclerosis, which increases the risk of cerebral in-farction. Oxidative stress response results in the loss of protein and lipid biological functions in the body, damages the cell structure and produces a large number of oxidative products (MDA and ox-LDL)25. During ACI, the endogenous oxida-tive mechanism of local tissues will be activat-ed, which is manifested as the up-regulation of antioxidant enzymes (GPX and SOD). However, the excessive production of oxygen radicals can weaken the antioxidant capacity of the body. In TUDCA group, the content of oxidative products (MDA and ox-LDL) was decreased, but the levels of antioxidant enzymes (GPX and SOD) were in-creased compared with those in ACI group (Fig-ure 4), suggesting that TUDCA can alleviate the damage caused by ACI oxidative stress through its antioxidant effect.Researchers showed that the up-regulation of Nrf2 can positively regulate the expression of VLDLR in alcoholic liver disease, thereby leading to the accumulation of VLDLR in liver cells26. Some studies have also demonstrated that Nrf2 gene silencing can reduce the VLDLR expression. NF-κB is a classical pro-inflammato-ry factor, which has been proved to be activated by the activation of Nrf2, thereby mediating the production of inflammatory factors and inducing the inflammatory cascade27. The expressions of Nrf2, NF-κB, and VLDLR in TUDCA group

Figure 5. (a) The contents of Nrf2, VLDLR, NF-κB, caspase-3 and Bax proteins in brain tissues in each experimental group are detected via Western blotting, and the relative gray value of each protein (b) is measured and analyzed statistically, showing statistically significant differences (*p < 0.05).

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were all decreased (Figure 5), so it is speculated that the regulatory effects of TUDCA on inflam-mation and lipid are at least partially related to the Nrf2 signaling pathway. It has been confirmed

via the application of caspase inhibitors and ge-netically-engineered mice that caspase-mediated apoptosis plays an important role in the ischemic injury-induced excessive neuronal apoptosis28. Scholars have proved that TUDCA can inhibit the caspase cascade, thus inhibiting the activation of caspase-329. Some researchers have also showed that TUDCA inhibits the liver cell apoptosis through inhibiting mitochondrial depolarization and production of reactive oxygen30. Besides, Bax is a pro-apoptotic gene in the Bcl-2 gene family, which can cause apoptosis through combining with the mitochondrial outer membrane. In this study, the expressions of caspase-3 and Bax in TUDCA group were down-regulated (Figure 5). Therefore, it is reasonable to speculate that TUD-CA reduces the degree of ACI through inhibit-ing caspase apoptosis pathway and mitochondrial apoptosis pathway.

Conclusions

We found that TUDCA can alleviate the ACI-induced neurological impairment in rats through reducing lipid peroxidation, inflam-matory response, and apoptosis. Its underlying mechanism may be that TUDCA can negatively regulate Nrf2 signaling pathway, down-regulate the expressions of VLDLR, and NF-κB proteins and expressions of apoptotic proteins (Bax and caspase-3). Our results provide new direction and theoretical guidance for the clinical development of ACI and the drug development.

Conflict of InterestThe Authors declare that they have no conflict of interests.

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