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IMR Press / JIN / Volume 21 / Issue 6 / DOI: 10.31083/j.jin2106160
Open Access Original Research
Is Matrix Metalloproteinase-9 Associated with Post-Stroke Cognitive Impairment or Dementia?
Jianhua Zhao1,†,*Fangli Yang1,†Xue Peng2,†Qing Li1Fan Wang1Qiong Li1Sibei Ji1
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1 Department of Neurology, First Affiliated Hospital of Xinxiang Medical University, Henan Joint International Research Laboratory of Neurorestoratology for Senile Dementia, Henan Key Laboratory of Neurorestoratology, 453100 Xinxiang, Henan, China
2 Department of Neurology, the Sixth Hospital of Wuhan, Affiliated Hospital to Jianghan University, 430015 Wuhan, Hubei, China
3 Department of Medical Record Management, First Affiliated Hospital of Xinxiang Medical University, 453100 Xinxiang, Henan, China
4 Department of Imaging, First Affiliated Hospital of Xinxiang Medical University, 453100 Xinxiang, Henan, China
5 Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
*Correspondence: 051092@xxmu.edu.cn (Jianhua Zhao)
These authors contributed equally.
J. Integr. Neurosci. 2022, 21(6), 160; https://doi.org/10.31083/j.jin2106160
Submitted: 10 June 2022 | Revised: 2 August 2022 | Accepted: 2 August 2022 | Published: 22 September 2022
This is an open access article under the CC BY 4.0 license.
Abstract

Background: Matrix metalloproteinase-9 (MMP-9) is a significant protease required for synaptic plasticity, learning, and memory. Yet, the role of MMP-9 in the occurrence and development of cognitive decline after ischemic stroke is not fully understood. In this study, we used clinical data experiments to further investigate whether MMP-9 and genetic polymorphism are associated with post-stroke cognitive impairment or dementia (PSCID). Materials and Methods: A total of 148 patients with PSCID confirmed by the Montreal Cognitive Assessment (MoCA) 3 months after onset (PSCID group) were included in the study. The MMP-9 rs3918242 polymorphisms were analyzed using polymerase chain reaction coupled with restriction fragment length polymorphism, and the serum level of MMP-9 was measured using enzyme-linked immunosorbent assay (ELISA). The same manipulations have been done on 169 ischemic stroke patients without cognitive impairment (NCI group) and 150 normal controls (NC group). Results: The expression level of serum MMP-9 in the PSCID group and NCI group was higher compared to the NC group, and the levels in the PSCID group were higher than that in the NCI group (all p $<$ 0.05). Diabetes mellitus, hyperhomocysteinemia, and increased serum MMP-9 levels were the main risk factors of cognitive impairment after ischemic stroke. The serum level of MMP-9 was negatively correlated with the MoCA score, including visual-spatial executive, naming, attention, language, and delayed recall. Genetic polymorphism showed that TC genotype with MMP-9 rs3918242 and CC genotype were associated with a significantly increased risk of PSCID; moreover, the TC genotype significantly increased the risk of cognitive impairment. In the TCCC genotype of MMP-9 rs3918242, diabetes mellitus and hyperhomocysteinemia were associated with the increased risk of PSCID; also, hyperhomocysteinemia could increase the risk of cognitive impairment. Conclusions: MMP-9 level and MMP-9 rs3918242 polymorphism have an important role in the occurrence and development of post-stroke cognitive impairment or dementia (PSCID).

Keywords
ischemic stroke
post-stroke cognitive impairment or dementia (PSCID)
matrix metalloprotenase 9
genetic polymorphism
cognitive function
1. Introduction

Vascular cognitive impairment (VCI) is a heterogeneous disease that involves cognitive decline characterized by disturbance of frontal or executive dysfunction [1]. VCI is usually a consequence of cerebrovascular disorders (cerebral infarction, cerebral hemorrhage, chronic cerebral hypoperfusion) and their risk factors (hypertension, diabetes, hyperlipemia, hyperhomocysteinemia, etc.) [1, 2]. Post-stroke cognitive impairment or dementia (PSCID; VCI after cerebral ischemic stroke) is very common and can affect different cognitive domains. Executive functions are the most commonly affected funtions [3]. Impairment in cognitive, especially executive functions, commonly appears within 3 months after stroke. According to various hospital-based studies [4, 5, 6], the prevalence of PSCID varies from 11.6% to 56.3%. Therefore, neurorestoration of cognitive impairment has gained increasing interest among researchers [7, 8, 9].

Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family with 26 extracellular and intracellular matrix-degrading enzymes that regulate many zinc-binding proteolytic enzymes physiological processes, including activation of growth factors, tumor growth and metastasis, cleavage of zymogens, and remodeling of the extracellular matrix [7, 8]. MMP-9 is mainly produced by neurons, and its expression and activity have been detected in adult brain structure, such as the hippocampus, cortex, striatum, and cerebellum [9, 10]. In neurons, the MMP-9 expression is induced by neuronal activity under both physiological and pathological conditions, such as stroke [11, 12] and epilepsy [13]. Abnormalities and typically excessive MMP-9 gene expression have been associated with several central nervous system diseases [14, 15, 16]. Moreover, MMP-9 is involved in blood-brain barrier destruction [17], inflammatory reaction [18], atherosclerosis, and ischemic stroke [19], as well as in synaptic plasticity [20], learning, and memory [21]. Yet, the role of MMP-9 in the occurrence and development of PSCID is not fully understood.

So far, only a few studies reported on the role of MMP-9 in VCI and its progression to dementia [22, 23, 24]. Even some studies have not found correlation with MMP-9 polymorphism and cognition [25, 26, 27]. In this study, we use enzyme-linked immunosorbent assay (ELISA) to detect serum MMP-9 levels and their association with PSCID. Furthermore, genotyping assays were performed to determine the relationship between the MMP-9 rs3918242 polymorphism and susceptibility of PSCID in a Chinese population.

2. Materials and Methods
2.1 Study Population

A total of 2351 acute ischemic stroke patients consecutively enrolled in the Department of Neurology, the First Affiliated Hospital of Xinxiang Medical University, Henan, China, between June 2013 and September 2018 were screened. The main inclusion criteria were: (1) ischemic stroke confirmed by imaging (computed tomography (CT) or magnetic resonance imaging (MRI)) within 72 hours of symptom onset, according to the criteria of the 10th Edition of International Classification of Diseases (ICD-10); (2) unrelated Han Chinese people and a Mini-Mental State Examination (MMSE) score of illiterate $>$ 17, primary $>$ 20, and secondary or higher $>$ 24 out of 30. MMSE scores were based on the cultural level of the subjects. Patients with brain tumors, brain trauma, thyroid dysfunctions, alcoholism, severe medical conditions, or neurological condition with consciousness were excluded. Also, those with severe stroke with NIHSS (National Institute of Health stroke scale) $>$ 25 or patients unable to complete assessment were excluded from this study. Exclusion criteria and the exact number of patients for each study stage are shown in Fig. 1.

Fig. 1.

Flow chart of participants’ selection.

After the onset of 3 months [28], 583 patients underwent a follow-up evaluation, and 486 patients were assessed with global cognitive functions by Changsha version of the Montreal Cognitive Assessment (MoCA-CS) [29] and according to the flow chart [30, 31]. Yu K.H’s protocol was referred (Fig. 1) [32]. Age- and gender-matched cognitively normal patients after ischemic stroke (MoCA $\geq$ 26) were used as a control. In addition, age- and gender-matched 150 healthy control subjects unrelated Han people with no ischemic stroke and cognitive dysfunction were randomly selected from the outpatients for health check-ups.

2.2 Cognitive Screening Measures

All subjects were evaluated based on MMSE and MoCA (Fig. 1). MoCA was scored based on a 30-point scale with 7 cognitive subtests: visual-spatial executive, naming, attention, language, abstraction, delayed recall, and orientation. The MoCA test was conducted to rectify the educational level of the bias correction, and subjects with education $<$ 12 years were given an additional 1 score on their test results. A higher score indicates the better cognitive function, and $\geq$ 26 were divided into normal [33]. MoCA-CS in China was used in this study [29].

Unified and standardized survey questionnaire terminologies were used in a quiet environment without interferences. The neuropsychological assessments for each patient were completed by one psychological surveyor on the same day.

2.3 DNA Extraction

The blood samples were collected during cognitive assessment between three months and half a year after stroke. And the blood sample were collected in EDTA-containing tubes and separated the serum immediately and frozen at –80 ${}^{\circ}{}$ C until further use. Genomic DNA was isolated using the TIANamp Blood DNA Kit (Tiangen, Beijing, China) according to the kit’s protocol. The lab technician performing the test was blinded to all patients and clinical information, and all lab work was carried out in the First Affiliated Hospital of Xinxiang Medical University.

2.4 Genotyping Assays

The MMP-9 rs3918242 polymorphisms were performed by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism, as described in our previous studies [19].

2.5 MMP-9 Level in Serum

The collection, processing and storage of blood sample are same as DNA extraction. The process of measuring MMP-9 is according to the MMP-9 ELISA Kit’s (Wuhan Fine Biotech Co., Ltd.; Wuhan; China) protocol.

2.6 Statistical Analysis

SPSS 24.0 (SPSS Inc., Chicago, IL, USA) was used for statistical processing. The measurement data in the results were expressed as mean $\pm{}$ SD, and the count data were expressed as a percentage. Analysis of variance and two independent samples t-test were used for normally distributed data; rank-sum test was used for non-normally distributed data, and the $\chi{}$ ${}^{2}$ test was used for the count data to compare the statistical differences of demographic and clinical data between groups. Spearman correlation analysis was used to analyze the correlation between MMP-9 and MoCA score. Logistic regression analysis was used to analyze the risk factors of PSCID by estimating the odds ratio (OR) and 95% confidence interval (95% CI). The Hardy-Weinberg equilibrium of MMP-9 rs3918242 genotype was tested by Chi-square ( $\chi{}$ ${}^{2}$ ) goodness-of-fit test. The associations between the MMP-9 rs3918242 polymorphisms and PSCID risk were determined by logistic regression. The major homozygous genotype of MMP-9 rs3918242 was used as a reference, two-tailed, and p $<$ 0.05 was considered statistically significant.

3. Results
3.1 Comparison of Baseline Demographic Features, Vascular Risk Factors, and Location of Cerebral Infarction in the Three Groups

There were significant differences in diabetes, hyperhomocysteinemia, serum MMP-9 levels, and MoCA scores between the three groups (all p $<$ 0.05). Compared with the NC group, the serum MMP-9 expression level in the PSCI group and the NCI group was significantly increased; yet, the MMP-9 expression level in the PSCI group was significantly higher than that in the NCI group (all p $<$ 0.05). Moreover, the MoCA scores of the PSCI group and the NCI group were significantly reduced compared with the NC group, and the MoCA scores of the PSCI group were significantly lower than that of the NCI group (p $<$ 0.05) (Table 1).

Table 1.Comparison of baseline demographic features, vascular risk factors, and location of cerebral infarction in the three groups.
 Variables Groups F/Z/χ ${}^{2}$ p PSCID NCI NC (n = 148) (n = 169) (n = 150) Age [years] $\leq$ 60 73 (49.32) 88 (52.07) 73 (48.67) $>$ 60 75 (50.68) 81 (47.93) 77 (51.33) 0.42 0.81 Gender Females 98 (66.22) 107 (63.31) 100 (66.67) Males 50 (33.78) 62 (36.69) 50 (33.33) 0.47 0.79 Educations [years] $\leq$ 24 102 (68.92) 118 (69.82) 102 (68.00) $>$ 24 46 (31.08) 51 (30.18) 48 (32.00) 0.12 0.94 NIHSS ${}^{†}$ 8.59 $\pm$ 5.27 8.37 $\pm$ 5.47 - 0.34 0.56 Hypertension No 52 (35.14) 63 (37.28) 54 (36.00) Yes 96 (64.86) 106 (62.72) 96 (64.00) 0.16 0.92 Diabetes mellitus No 94 (63.51) 129 (76.33) 113 (75.33) Yes 54 (36.49) 40 (23.67) 37 (24.67) 7.68 0.02 Hyperlipidemia No 128 (86.49) 154 (91.12) 141 (94.00) Yes 20 (13.51) 15 (8.88) 9 (6.00) 3.49 0.18 Hyperhomocysteinemia No 62 (41.89) 93 (55.03) 86 (57.33) Yes 86 (58.11) 76 (44.97) 64 (42.67) 8.36 0.02 Tobacco smoking Never 95 (64.19) 108 (63.91) 96 (64.00) Yes 53 (35.81) 61 (36.09) 54 (36.00) 0.00 1.00 Alcohol intake Never 78 (52.70) 102 (60.36) 90 (60.00) Yes 70 (47.30) 67 (39.64) 60 (40.00) 2.33 0.31 TOAST classification Large-artery atherosclerosis 52 (35.14) 59 (34.91) - Small vessel occlusion 64 (43.24) 71 (42.01) - Cardioembolism 10 (6.76) 12 (7.10) - Other determined etiology 13 (8.78) 16 (9.47) - Undetermined etiology 9 (6.08) 11 (6.51) - 0.11 1.00 MMP-9 (mg/dL) ${}^{†}$ 299.07 $\pm$ 101.67* ${}^{\#}$ 266.56 $\pm$ 98.23* 197.75 $\pm$ 86.92 88.14 $<$ 0.001 MoCA ${}^{†}$ 20.27 $\pm$ 3.54* ${}^{\#}$ 28.62 $\pm$ 1.24* 29.50 $\pm$ 0.90 337.74 $<$ 0.001 *p $<$ 0.05 compared with NC group; #p $<$ 0.05 compared with NCI group. ${}^{\textdagger{}}$ Use of the rank-sum test. BMI, Body mass index; NIHSS, National Institutes of Health Stroke Scale; TOAST, Trial of Org 10172 in Acute Stroke Treatment.
3.2 Logistic Regression Analysis of PSCID

With diabetes mellitus, when hyperhomocysteinemia and serum MMP-9 level were used as independent variables, and PSCID as dependent variables, logistic regression analysis showed that diabetes mellitus, hyperhomocysteinemia, and elevated serum MMP-9 level were the main risk factors of PSCID (OR = 1.77, 95% CI: 1.07–2.93; OR = 1.88, 95% CI: 1.18–3.56; OR = 1.01, 95% CI: 1.01–1.02) (Table 2).

Table 2.Logistic regression analysis of PSCID.
 Variables Beta value Standard error Wald value OR 95% CI p Diabetes mellitus (%) 0.63 0.24 4.91 1.77 1.07–2.93 0.03 Hyperhomocysteinemia (%) 0.57 0.26 7.10 1.88 1.18–3.56 0.01 MMP-9 (mg/dL) 0.01 0.0003 51.31 1.01 1.01–1.02 $<$ 0.001
3.3 Correlation between Serum MMP-9 Level and MoCA Score in Patients with PSCID

The Spearman correlation analysis showed that the serum MMP-9 level was negatively correlated with the total score of MoCA scale in patients with PSCID (p $<$ 0.05), and the serum MMP-9 level was negatively correlated with visual-spatial executive, naming, attention, language and delayed recall (p $<$ 0.05), but not with abstraction and orientation (p $>$ 0.05) (Table 3).

Table 3.Correlation between serum MMP-9 level and MoCA score in patients with PSCID.
 Variables Fraction (mean $\pm$ SD) MMP-9 (ng/mL) r p Visual spatial executive 2.74 $\pm$ 1.46 –0.401 0.000 Naming 2.59 $\pm$ 0.58 –0.230 0.005 Attention 4.27 $\pm$ 1.41 –0.470 0.000 language 1.99 $\pm$ 0.76 –0.255 0.002 Abstraction 1.18 $\pm$ 0.81 –0.006 0.944 Delayed recall 2.66 $\pm$ 1.09 –0.165 0.045 Orientation 4.84 $\pm$ 0.789 –0.130 0.116 Total 20.27 $\pm$ 3.54 –0.517 0.000
3.4 The Polymorphism Distribution of MMP-9 rs3918242 in Each Group

The genotype frequency of MMP-9 rs3918242 in the NC group conformed to Hardy-Weinberg equilibrium (p $>$ 0.05), while the genotype frequency of MMP-9 rs3918242 in the PSCID group and the NCI group did not (p $<$ 0.05). The $\chi{}$ ${}^{2}$ test showed that the genotypes of the three groups were significantly different ( $\chi{}$ ${}^{2}$ = 14.57, p $<$ 0.05) (Table 4).

Table 4.The polymorphism distribution of MMP-9 rs3918242 in each group.
 MMP-9 rs3918242 Genotype % Allele % Hardy-Weinberg equilibrium TT TC CC T C $\chi$ ${}^{2}$ p PSCID (%) 77 (52.03) 45 (30.40) 26 (17.57) 199 (67.23) 97 (32.77) 14.22 0.001 NCI (%) 109 (64.50) 43 (25.44) 17 (10.06) 261 (77.22) 77 (22.78) 12.95 0.002 NC (%) 102 (68.00) 40 (26.67) 8 (5.33) 244 (81.33) 56 (18.67) 2.22 0.329 $\chi$ ${}^{2}$ 14.57 p 0.006
3.5 Association between MMP-9 rs3918242 and Risk of PSCID

The logistic regression analysis showed TC genotype carrying MMP-9 rs3918242 (OR = 3.91, 95% CI: 1.63–9.38) and CC genotype (OR = 2.79, 95% CI: 1.12–7.00) were associated with a significantly increased risk of PSCID (p $<$ 0.05). Also, in PSCID and NCI groups, TC genotype carrying MMP-9 rs3918242 (OR = 2.03, 95% CI: 1.02–4.05) significantly increased the risk of PSCID using TT genotype as a control (p $<$ 0.05) (Table 5).

Table 5.Association between MMP-9 gene rs3918242 and risk of VCI after ischemic stroke.
 MMP-9 rs3918242 Groups OR (95% CI) * p Groups OR (95% CI) * p PSCID (%) NCI (%) PSCID (%) NC (%) TT 77 (52.03) 102 (68.00) 1.0 (Ref.) - 77 (52.03) 109 (64.50) 1.0 (Ref.) - TC 45 (30.40) 40 (26.67) 3.91 (1.63–9.38) 0.002 45 (30.40) 43 (25.44) 2.03 (1.02–4.05) 0.044 CC 26 (17.57) 8 (5.33) 2.79 (1.12–7.00) 0.028 26 (17.57) 17 (10.06) 1.51 (0.71–3.22) 0.282 * Adjusted for Diabetes mellitus, Hyperhomocysteinemia.
3.6 Association between MMP-9 rs3918242 and Risk of PSCID Stratified by Demographic Characteristics

The relationship between MMP-9 rs3918242 and PSCID risk of was further analyzed and stratified by demographic characteristics such as diabetes mellitus and hyperhomocysteinemia (Table 6). The logistic regression analysis showed that diabetes mellitus and hyperhomocysteinemia in the TCCC genotype of MMP-9 rs3918242 were associated with increased risk of PSCID (OR = 1.26, 95% CI: 1.14–2.66; OR = 1.24, 95% CI: 1.02–2.69). In addition, hyperhomocysteinemia increased the risk of PSCID (OR = 1.19, 95% CI: 1.10–2.38).

Table 6.Association between MMP-9 rs3918242 and risk of PSCID stratified by demographic characteristics.
 Variables PSCID NC OR (95% CI) p PSCID NCI OR (95% CI) p TT TC+CC TT TC+CC TT TC+CC TT TC+CC Diabetes mellitus No 51 43 86 43 1.65 (0.87–3.14) 0.13 51 43 73 40 1.59 (0.84–3.02) 0.06 Yes 26 28 23 17 1.26 (1.14–2.66) 0.01 26 28 29 8 1.69 (0.90–3.56) 0.37 Hyperhomocysteinemia No 47 15 53 40 1.51 (0.71–3.16) 0.27 47 15 58 28 2.37 (0.86–4.82) 0.22 Yes 30 56 56 20 1.24 (1.02–2.69) 0.00 30 56 44 20 1.19 (1.10–2.38) 0.00
4. Discussion

Our data suggested that TC and CC genotypes of MMP-9 rs3918242 polymorphism were associated with a significantly increased risk of PSCID in a Han Chinese population, further suggesting an association between rs3918242 variation in the MMP-9 gene and PSCID. We also found that the TC genotype of rs3918242 increased the risk of PSCID compared to the TT genotype. Our results support the hypothesis that MMP-9 gene polymorphisms are associated with PSCID. In addition, our data suggested that diabetes mellitus, hyperhomocysteinemia, and MMP-9 serum levels were associated with PSCID. However, there was no significant association between PSCID and other demographic features (such as age, gender, hypertension, hyperlipidemia, tobacco smoking, alcohol intake, as well as TOAST classification). Moreover, MMP-9 in the serum was negatively correlated with MoCA score, especially visual-spatial executive, naming, attention, language, and delayed recall.

Numerous studies have investigated the relationships between MMP-9 gene polymorphisms and ischemic stroke. However, these results remain debatable. A meta-analysis showed that MMP-9 (-1562C/T) gene polymorphism was not associated with ischemic stroke [34]. In addition, Wang et al. [35] showed that the MMP-9 gene rs3918242 and rs17577 polymorphisms are not significantly correlated with ischemic stroke risk. Contrary, another meta-analysis suggested that MMP-9 (-1562C/T) polymorphisms might be a risk factor for ischemic stroke [36]. Also, several recent studies have reported the significant association between MMP-9 and cerebral ischemic stroke [15, 37, 38]. Gao et al. [37] showed that the rs3787268 locus in the MMP-9 gene might increase the risk of ischemic stroke in a southern Chinese Han population. Moreover, Hao and colleagues [16] conducted a similar study with 317 patients and found that MMP-9 rs3918242 polymorphism is correlated with an elevated risk of ischemic stroke. In addition, Li et al. [15] observed a significant association between the MMP-9 rs3918242 polymorphism and the risk of ischemic stroke, which is consistent with our previous study [19].

Preclinical studies showed that MMP-9 undergoes high expression and activation in brain tissue after a major complication of acute and chronic stroke [24, 39], suggesting that MMP-9 may participate in the acute brain injury and edema resulting from neuroinflammation [40, 41, 42]. On the other hand, secondary disruption to the deep white matter, the blood-brain barrier (BBB), and demyelination at the cerebral brain [24, 39, 43, 44], have an important role in the pathogenesis of vascular cognitive impairment and vascular dementia [22, 43]. Some clinical trials have reported elevated MMP-9 levels in the serum of patients with stroke [45, 46]. The high levels of MMP-9 in acute ischemic stroke confirm the involvement of this metalloproteinase in the regulation of inflammation in stroke [47, 48]. Meanwhile, animal studies suggested that MMP-9 has a role in cognitive functions, especially learning and memory [21, 49, 50, 51]. Therefore, the expression level of MMP-9 can be dispensable for PSCID. An increasing number of reports indicate that MMP-9 levels are in cerebrospinal fluid [22, 39, 41, 52] and peripheral blood [23, 53] of patients with PSCID. Furthermore, our data showed higher serum MMP-9 levels in patients with PSCID compared to normal controls and patients without cognitive impairment, which is consistent with the previous study. Furthermore, the serum level of MMP-9 was negatively correlated with the MoCA score, especially visual-spatial executive, naming, attention, language, and delayed recall.

This study has a few limitations. It has a relatively small sample size. Also, changes in MMP-9 expression at different development periods of PSCID were not analyzed. It is widely accepted that a large sample size is a key issue in genetic polymorphisms association studies of complex traits generally and cognitive dysfunction specifically [54]. In addition, it is unlikely that a polymorphism in a single gene would have a profound effect on the risk of PSCID which may be caused by many risk factors. Furthermore, the distribution and expression of MMP-9 in the pathogenesis and progression of post-stroke cognitive impairment or dementia are dynamic. According to the previous studies [21, 22, 53, 55], MMP-9 expression levels are associated with PSCID, although they are not exactly equal in serum, cerebrospinal fluid, and brain tissue after BBB disruption. So, multi-center, multi-source, large sample size studies investigating PSCID and dynamic monitoring of MMP-9 are needed to further confirm these findings.

5. Conclusions

Our data and previous studies suggest that MMP-9 level and MMP-9 rs3918242 gene polymorphism are associated with post-stroke cognitive impairment or dementia (PSCID). MMP-9 has a role in the development of post-stroke cognitive impairment or dementia (PSCID); yet, the mechanisms of action need to be further explored.

Author Contributions

JZhao and SL designed the study and wrote the protocol. FY and XP enrolled the patients and wrote the paper. QingL, FW and ZX were in charge of follow-up of patients. RC, DJ, JZhang, MW and QiongL interpreted the patients’ image data. SJ and SL help in manuscript revision and interpretation of the results.

Ethics Approval and Consent to Participate

The study was approved by the Ethics Committee of the first affilicated hospital of Xinxiang Medical University. The ethical statement No. is EC-022-016. All subjects gave written informed consent in accordance with the Declaration of Helsinki.

Acknowledgment

Not applicable.

Funding

Natural Science Foundation of Henan Province (182300410389); Joint construction project of Henan Medical Science and technology research plan (LHGJ20190437); Xinxiang Medical University Scientific Research Innovation Support Program Project (YJSCX202185Y).

Conflict of Interest

The authors declare no conflict of interest.

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