Serum PPARγ level and PPARγ gene polymorphism as well as severity and prognosis of brain injury in patients with arteriosclotic cerebral infarction.

The aim of the study was to study the serum peroxisome proliferator-activated receptor gamma (PPARγ) level and PPARγ gene polymorphism as well as the severity and prognosis of brain injury in patients with arteriosclotic cerebral infarction (ACI). A total of 246 ACI patients presenting at the Department of Neurology of Zengcheng District People's Hospital of Guangzhou between April 2009 and July 2015 were selected as the case group, and 382 control subjects were enrolled as the control group. The hepatic and renal functions and homocysteine (Hcy) expression levels were measured. Enzyme-linked immunosorbent assay (ELISA) kit was used to detect the serum PPARγ levels of the ACI patients. Polymerase chain reaction-restriction fragment length polymorphism method was applied to measure the PPARγ gene polymorphism. The proportions of hypertension patients, diabetes patients and smoking people in the case group were significantly higher than those in the control group. The levels of cholesterol and fasting blood glucose in the case group were elevated obviously compared with those in the control group. The levels of indexes related to the hepatic function and renal function in the case group were remarkably higher than those in the control group. The serum PPARγ levels were increased progressively at acute stage. The distribution frequencies of PPARγ genotypes CC, CT and TT in the case group were higher than those in the control group; compared with that in the control group, the proportion of C allele in the case group was raised obviously, while that of T allele was significantly decreased. The serum PPARγ level has a close correlation with the PPARγ gene polymorphism in ACI patients, and PPARγ is also remarkably related to the severity of brain injury; therefore, PPARγ has great significance in the diagnosis and treatment of cerebral infarction.

Introduction

As a type of ligand-activated transcriptional factor of nuclear receptor that is correlated with metabolic regulation, peroxisome proliferator-activated receptor gamma (PPARγ) plays a vital role in controlling the storage and release of fats, glucose and lipid metabolism and energy metabolism, regulating insulin resistance, blood glucose, cell growth and differentiation and vascular inflammation reaction as well as influencing the pathological processes of vascular sclerosis (1–3). Based on the PPARγ expression in the brain neuron cells, PPARγ molecules may be released to the blood circulation when the brain tissue cells are injured (4, 5). However, whether the serum PPARγ level is associated with the severity of arteriosclotic cerebral infarction (ACI) and whether such an association is further closely related to the nucleotide polymorphism of PPARγ gene still need to be determined by more experiments.

As a result, this research aimed to provide a basis and direction for assessing the risk of cerebrovascular disease among the ACI patients by detecting their serum PPARγ level and PPARγ gene polymorphism in clinical practices.

Materials and methods

Information

General information

In this study, 246 ACI patients presenting at the Department of Neurology of Zengcheng District People's Hospital of Guangzhou (Guangzhou, China) from April 2009 to July 2015 were selected as the case group, and 382 subjects who were excluded of cerebral infarction through health examination were enrolled as the control group. The 246 patients in the case group were comprised of 182 males and 64 females, with an average age of (59.7±12.1) years. Of the 382 participants in the control group, there were 226 males and 156 females, with an average age of (56.1±10.8) years.

The study was approved by the Ethics Committee of Zengcheng District People's Hospital of Guangzhou and written informed consents were signed by the patients and/or guardians.

Experimental methods

General indexes

The general conditions of the participants were recorded, including age, sex, body mass index and history of hypertension, diabetes and smoking.

Blood routine tests

Fasting venous blood (5 ml) of all the participants was withdrawn in the morning to measure the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and fasting blood glucose. The levels and differences of indexes in the case group and the control group were recorded and compared.

Hepatic function measurement

The body mass indexes as well as the levels of indexes related to hepatic function, namely, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in the case group and the control group were measured and recorded, and the differences in the data were compared.

Renal function measurement

The data of indexes related to the renal function [blood urea nitrogen (BUN), serum creatinine (Scr) and uric acid (UA)] in the case group and the control group were measured, and the differences between the two groups were recorded.

Homocysteine (Hcy) level measurement

Hcy is a vital metabolic intermediate of sulfur-containing amino acid in the human body, which may be an independent risk factor for the onset of atherosclerosis and other cardiovascular diseases. The Hcy levels in both the case and control groups were measured, and the differences were compared.

PPARγ level measurement

Human PPARγ enzyme-linked immunosorbent assay (ELISA) kit was applied to detect the serum PPARγ levels in ACI patients three times, i.e., at acute stage (within 3–6 h), 48–72 h after ACI attack and one week after attack.

Detection of PPARγ gene

The blood samples of the ACI patients were collected to extract the genomic DNA (gDNA) of peripheral blood leucocyte and detect the PPARγ gene. The peripheral venous blood of the research subjects was withdrawn to extract the DNA. Later, polymerase chain reaction and gel electrophoresis were performed to detect the PPARγ gene. The upstream primer for the PPARγ gene was 5′-TGAATGTGAAGCCCATTGAA-3′, and the downstream primer was 5′-GAGCGGGTGAAGAAGACTCATGT-3′.

Statistical analysis

The experimental data were presented as mean ± standard deviation (mean ± SD), and the experimental results were analyzed using Statistical Product and Service Solutions (SPSS) 17.0 software. The analysis of variance or t-test was conducted for data analysis and the post hoc test was LSD test. P<0.05 suggested that the difference between the two groups was statistically significant.

Results

Measurement results of general indexes

The general conditions of the participants were recorded; it was known from Table I that in the case group, the proportion of hypertension patients was 41.82%, the proportion of diabetes patients was 26.83%, and that the proportion of smoking people was 30.08%; the proportions of hypertension patients, diabetes patients and smoking people were 21.47, 14.14 and 14.92%, respectively, in the control group. It could be seen that cerebral infarction had a certain correlation with hypertension, diabetes and smoking.

Table I.

Comparison of general information between the case group and the control group.

Group	Hypertension patient/case (%)	Diabetes patient/case (%)	Smoking people/case (%)
Case group	103 (41.82%)	66 (26.83%)	74 (30.08%)
Control group	82 (21.47%)	54 (14.14%)	57 (14.92%)

Results of blood routine tests

As shown in Fig. 1, the fasting venous blood of all the participants was withdrawn in the morning to measure the TC, TG, LDL-C and fasting blood glucose. The levels of TC, TG, LDL-C and fasting blood glucose in the case group were obviously higher than those in the control group.

Figure 1.

Results of TC, TG, LDL-C and fasting blood glucose levels in the case group and the control group. Compared with that in the case group, *p<0.05, **p<0.01.

Results of hepatic function measurement

The measurement results of body mass indexes as well as the levels of indexes related to the hepatic function (ALT and AST) in the case group and the control group are shown in Fig. 2. The body mass index in the case group was slightly higher than that in the control group but without significant difference. The levels of hepatic function indexes ALT and AST in the case group were significantly elevated compared with those in the control group. Thus, liver injury of the ACI patients was severe.

Figure 2.

Results of body mass indexes as well as levels of hepatic function indexes ALT and AST in the case group and the control group. Compared with that in the case group, **p<0.01.

Results of renal function measurement

The levels of indexes related to the renal function, i.e., BUN, Scr and UA, in the case group and the control group were measured and recorded (Fig. 3). It was indicated that the levels of BUN, Scr and UA in the case group were significantly higher than those in the control group. Therefore, the kidney injury of the ACI patients was severe.

Figure 3.

Results of expression levels of renal function-related indexes BUN, Scr and UA in the case group and the control group. Compared with that in the case group, **p<0.01.

Results of Hcy level measurement

Compared with that in the control group, the Hcy level in the case group was increased significantly (Fig. 4). Patients with a high Hcy level were vulnerable to thrombosis and cardiovascular diseases; thus, there was a close relationship between the ACI patients and cardiovascular diseases.

Figure 4.

Expression results of Hcy levels in the case group and the control group. Compared with that in the case group, **p<0.01.

Results of PPARγ level measurement

Human PPARγ ELISA kit was used to detect the serum PPARγ levels of the ACI patients at acute stage (within 3–6 h), 48–72 h after ACI attack and one week after attack. As shown in Fig. 5, the serum PPARγ levels were increased progressively at the acute stage (3–6 h), 48–72 h after ACI attack and one week after attack.

Figure 5.

Results of serum PPARγ levels of ACI patientsat acute stage, 48–72 h after ACI attack and one week after attack.

Results of PPARγ gene detection

The results of genotype distribution allele frequencies in the case group and the control group are shown in Table II. In the case group, the distribution frequencies of PPARγ genotypes CC, CT and TT were 74.6, 22.3 and 2.6%, respectively, the C allele frequency was 87.3%, and the Tallele frequency was 14.1%. In the control group, the distribution frequencies of genotypes CC, CT and TT were 61.1, 37.6 and 3.1%, respectively, of which the C allele frequency was 78.6%, and the T allele frequency was 20.6%.

Table II.

PPARγ genotypes and allele distribution in the case group and the control group.

	Genotype frequency (%)			Diabetes patient (%)
Group	CC	CT	TT	C	T
Case group	74.6%	22.3%	2.6%	87.3%	14.1%
Control group	61.1%	37.6%	3.1%	78.6%	20.6%

Discussion

ACI is also known as atherothrombotic brain infarction (6). It may occur in all ages, particularly in the elderly; many lifestyle habits and diseases, such as high-fat diet, diabetes and smoking, can induce and accelerate ACI (7–9). With the development of the society, improvement of living standards and huge changes in lifestyle in recent years, ACI has become a disease that poses a serious threat to both life and health, and its incidence rate is on the increase annually (10–13). However, the pathogenesis of ACI also remains unclear due to currently imperfect diagnosis and treatment of ACI. Multiple factors, including inflammation, oxidative stress and apoptosis, influence the occurrence and progression of the disease. Therefore, it is imminent to identify safe and effective ACI treatment methods (14,15).

There are three types of PPAR family, namely, PPARα, PPARβ and PPARγ, which have different histologic manifestations and reflect varying physiological functions (16). PPAR is praised as the ‘the ultimate mystery of human health’, whose amazing effects on promoting human health, preventing diseases and treating various difficult and miscellaneous diseases are discovered by more and more scientific research and clinical trials (17). The PPAR molecules in the human body are a group of nucleoprotein receptors with transcription-regulating function, which consist of the subtypes PPARα, PPARβ and PPARγ; they are widely present in the nuclei of liver, fat, vessel wall and skeletal muscle (18). The activated PPARs can affect the manifestations of different genes in the body, act on the mechanisms of adjusting metabolism of glucose and lipid, ameliorating resistance, guarding against inflammation and preventing cell malignant transformation, and operate for a ‘stable’ state in the human body (19). PPARγ is a superfamily of nuclear receptors, which is involved in many regulation processes for cell functions and pathophysiology at the transcription level, including adipocyte differentiation, glucose and lipid metabolism, inflammatory reaction, atherosclerosis as well as cancer cell differentiation and formation; in addition to the treatment of diabetes, the PPARγ agonist may be applied to treat atherosclerosis, tumors and inflammatory diseases in the future (20).

In this study, 246 ACI patients presenting at the Department of Neurology of our hospital between April 2009 and July 2015 were selected as the case group, and 382 control subjects, who were excluded of cerebral infarction through health examination, were enrolled as the control group. The general information and blood routine of the two groups were recorded. The hepatic and renal functions and Hcy expression level were measured, and ELISA was utilized to detect the serum PPARγ levels of the ACI patients at acute stage, 48–72 h after ACI attack and one week after attack. Polymerase chain reaction-restriction fragment length polymorphism method was applied to measure the PPARγ gene polymorphism, and the results indicated that the proportion of hypertension patients, diabetes patients and smoking people in the case group were significantly higher than those in the control group. The results of blood routine tests showed that the levels of TC, TG, LDL-C and fasting blood glucose in the case group were evidently elevated compared with those in the control group. The levels of the hepatic function-related indexes ALT and AST as well as renal function-related indexes BUN, Scr and UA in the case group were significantly higher than those in the control group. Moreover, compared with that in the control group, the Hcy level in the case group was increased notably. It was revealed in the ELISA results that the serum PPARγ levels were increased progressively at acute stage, 48–72 h after ACI attack and one week after attack. The distribution frequencies of PPARγ genotypes CC, CT and TT in the case group were higher than those in the control group. Compared with that in the control group, the proportion of C allele in the case group was significangtly increased, while that of T allele was lowered significantly. In conclusion, the serum PPARγ level has a close association with the PPARγ gene polymorphism in ACI patients, and PPARγ is also markedly related to the severity of brain injury. Thus, PPARγ can play a crucial role in the diagnosis and treatment of ACI in the future.