Clinical characteristics
Patients enrolled in this study included 13 control individuals (Control group), 14 patients in the DM group, 16 patients in nonDM + AIS group and 14 patients in the DM + AIS group. Clinical characteristics are shown in Table 1. The rates of smoking and drinking were higher in the nonDM + AIS group than in the Control group. There was no significant difference in age, history of hypertension and coronary heart disease between the four groups. The NIHSS score between nonDM + AIS and DM + AIS was not significantly different. The plasma concentrations of HbA1c and FBG were higher in the diabetic group than in the non-diabetic group (Table 1). There was no significant difference between the four groups in the laboratory test results for LDL, triglycerides, cholesterol, white blood cells count, neutrophils and monocytes (Table 1).
Table 1
Characteristics of the patient groups as stratified by diabetes and ischemic stroke
|
Control
(n = 13)
|
DM
(n = 14)
|
nonDM + AIS
(n = 16)
|
DM + AIS
(n = 14)
|
P value
|
Age
|
65 ± 6
|
63 ± 8
|
68 ± 0
|
67 ± 6
|
0.33
|
Gender (male/female)
|
4/9
|
9/5
|
11/5†
|
12/2‡
|
0.03
|
Hypertension (%)
|
8(61.5)
|
11(78.6)
|
14(87.5)
|
12(85.7)
|
0.33
|
NIHSS
|
/
|
/
|
3 ± 3
|
3 ± 3
|
0.65
|
HbA1c
|
6.01 ± 0.45
|
7.98 ± 1.30*
|
5.85 ± 0.48
|
8.43 ± 1.79 ‡
|
< 0.01
|
FBG (mmol/L)
|
4.84 ± 0.42
|
7.57 ± 2.61*
|
5.22 ± 0.78
|
8.09 ± 2.13 ‡
|
< 0.01
|
TG (mmol/L)
|
1.72 ± 0.76
|
2.24 ± 1.82
|
1.61 ± 1.12
|
1.47 ± 0.54
|
0.34
|
TC (mmol/L)
|
4.66 ± 0.79
|
4.58 ± 1.42
|
4.77 ± 0.80
|
4.77 ± 0.94
|
0.82
|
LDL (mmol/L)
|
2.56 ± 0.67
|
2.64 ± 0.88
|
2.80 ± 0.68
|
3.09 ± 0.71
|
0.25
|
WBC ×109/L
|
6.45 ± 1.21
|
6.64 ± 1.43
|
7.63 ± 2.82
|
7.21 ± 1.80
|
0.36
|
Neutrophils ×109/L
|
4.00 ± 1.06
|
4.08 ± 1.14
|
4.99 ± 2.26
|
4.93 ± 1.80
|
0.24
|
Monocytes ×109/L
|
0.48 ± .18
|
0.46 ± 0.18
|
0.57 ± 0.21
|
0.49 ± 0.15
|
0.40
|
Data are presented as mean ± SD; P value shows comparison by ANOVA analysis between the four groups. *p < 0.05 Control vs. DM; †p < 0.05 AIS vs. DM; ‡p < 0.05 AIS vs. DM + AIS. AIS, acute ischemic stroke; DM, diabetes mellitus; FBG, fasting blood glucose; LDL, low-density lipoprotein; NIHSS, National Institute of Health Stroke Score; TC, total cholesterol; TG, triglyceride; WBC, total white blood cell count. |
SPMs secretion from ox-LDL-stimulated macrophages was enhanced in patients with AIS, while the ratios to LTB 4 were decreased
First, we compared the secretion ex vivo of lipid mediators (LMs) by macrophages from patients with AIS (nonDM + AIS and DM + AIS groups) and without AIS (Control and DM groups). The analysis showed that the levels of RvD1, RvD2, LXA4 and LTB4 were all increased in the patients with AIS (nonDM + AIS and DM + AIS groups) compared to non-AIS patients (Control and DM groups) under unstimulated conditions, whereas there was no significant difference in MaR1 (Fig. 1A). We then explored changes in LMs produced by macrophages stimulated with ox-LDL. Ox-LDL stimulation increased the secretion of both pro-inflammatory LTB4 and pro-resolving RvD1, RvD2, LXA4 and MaR1 by macrophages cultured 24 h ex vivo. (Fig. 1C).
The ratio between SPMs and LTB4 has been widely used as a marker reflecting the balance of resolution and inflammation.(17–19) We calculated the ratios between each SPM and LTB4 to evaluate the function of resolution in macrophages from the different patient groups, and there was no difference between the AIS and nonAIS groups under unstimulated conditions (Fig. 1B). However, after challenge with ox-LDL, the ratios RvD1/LTB4, RvD2/LTB4 and MaR1/LTB4 were significantly lower in AIS compared to nonAIS groups (Fig. 1D). The LXA4/LTB4 ratio in AIS groups was not statistically different compared to nonAIS groups, although there was a trend of decrease (Fig. 1D).
DM disturbed the balance of SPMs to LTB 4 and impaired resolution of inflammation in unstimulated macrophages from AIS patients
We then explored the influence of DM on levels of LMs in the culture medium of unstimulated macrophages. Interestingly, the ratios of SPMs to LTB4 were lower in the DM + AIS group than that in the AIS group. However, there was no significant difference between DM and the Control group (Fig. 2A).
To further explore the dysfunction of inflammation resolution, we assessed the levels of key SPM synthases in homogenates of macrophages by Western blot, including 5-LOX and 15-LOX-1, as well as the RvD2 receptor, GPR18. There was no significant difference in the levels of 5-LOX between the four patient groups. The levels of 15-LOX-1 and GPR18 were lower in the DM + AIS group than in the nonDM + AIS group, but there was no significant difference between control and DM group (Fig. 2B).
The inflammatory markers CD206 and iNOS were analysed by Western blotting in macrophage homogenates. The levels of the pro-inflammatory M1 marker iNOS was higher, while the levels of M2 marker CD206 was lower in DM + AIS compared to nonDM + AIS (Fig. 3C). To further evaluate the M2/M1 polarization, we calculated the ratio of CD206/iNOS, which was lower in DM + AIS than in nonDM + AIS (Fig. 2C). There was no difference between the DM and Control group (Fig. 2C).
The analysis of representative MAPK pathway markers p38 and NFκB pathway markers p65 exhibited similar results in that the levels of their phosphorylated forms (p-p38 and p-p65) and the ratios to their total levels (p-p38/p38 and p-p65/p65) were higher in DM + AIS than in nonDM + AIS (Fig. 2D). There was no difference between Control and DM with regard to these signal transduction markers nor in their ratios. There was no difference in the total levels of p38 and p65 between the four groups (Fig. 2D).
Ox-LDL stimulation exacerbated the disturbance in resolution function in macrophages from AIS patients with DM
We further explored the influence of DM on resolution function of macrophages stimulated with ox-LDL. The analysis revealed that the ratios RvD1/LTB4, RvD2/LTB4 and MaR1/LTB4 were lower in DM + AIS than in nonDM + AIS (Fig. 3A), consistent with the data for unstimulated macrophages. It is worth noting that ratios between SPMs and LTB4 were lower in DM + AIS than in DM, while there was no difference between nonDM + AIS and Control (Fig. 3A). The difference between DM + AIS and DM was not seen in unstimulated macrophages, indicating that ox-LDL exacerbated the impairment of resolution in macrophages from AIS patients with DM.
The data from analysis of SPM synthases were consistent with the findings from unstimulated macrophages. The levels of 15-LOX-1 were lower in DM + AIS than in DM (Fig. 3B), whereas there was no significant difference in 5-LOX between the four groups (Fig. 3B). The RvD2 receptor GPR18 was upregulated in DM + AIS compared to DM (Fig. 3B), indicating a possible compensatory mechanism against decreased RvD2 production. Similar changes of 15-LOX-1 and GPR18 were also seen between nonDM + AIS and Control (Fig. 3B).
Further analysis of the macrophage M1/M2 phenotype revealed that under stimulation with ox-LDL the levels of CD206 were lower and the levels of iNOS higher in DM + AIS compared to DM (Fig. 3C). The CD206/iNOS ratio was significantly lower in DM + AIS than in DM (Fig. 3C).
The phosphorylated forms of the signal transduction factors (p-p38 and p-p65) and the p-p38/p38 and p-65/p65 ratios were higher in DM + AIS than in DM (Fig. 3D). The increase in p-p65/p65 in DM + AIS was not seen in unstimulated macrophages, indicating further activation of the NFκB pathway by ox-LDL stimulation of the macrophages from AIS patients with DM.
RvD2 alleviates ox-LDL-induced inflammatory response in macrophages from AIS patients with DM
The above results demonstrated impaired resolution and excessive inflammation in macrophages of AIS patients with DM. Next, we analysed the effect of one of the SPMs in these macrophages. Ox-LDL stimulation significantly reduced the ratio of CD206/iNOS in macrophages from DM patients, indicating a polarization towards a pro-inflammatory phenotype. Treatment with RvD2 markedly reduced this polarization as shown by an increased CD206/iNOS ratio (Fig. 4A).
Moreover, the MAPK pathway markers p-p38 and ratio of p-p38/p-38, as well as NFκB pathway markers p-65 and ratio of p-p65/p65, were all increased by ox-LDL stimulation (Fig. 4B), and treatment with RvD2 significantly downregulated these markers (Fig. 4B).