3.1 Effect of activated B cells on myocardial fibrosis in mice with acute myocardial infarction
3.1.1. General situation of mice
After the AMI mice were fully awake, the mice showed with reduced activity and no obvious stimulus-response, while the mice in the Sham group generally responded normally. Ten mice in the AMI group died, including 2 mice in the 3 days and 1-week subgroups, and 3 mice in the 5-day and 2-week subgroups. No mice died in the Sham group. Gross specimen and pathological findings of mouse LAD after ligation were shown in figure 1.
3.1.2. The ratio of activated B cells in mouse myocardium, spleen, and peripheral blood
In myocardial tissue, the proportion of activated B cells in the AMI group was higher than that in the Sham group at 3 days, 5 days, and 1 week (P<0.05), no significant difference from the Sham group at 2 weeks (P>0.05),and the highest level was represented at 5 days subgroup (P<0.05) In the spleen and peripheral blood, the proportion of activated B cells in the AMI group was higher than that in the Sham group on 3 and 5 days(P<0.05), and the highest level was represented at 5 days subgroup (P<0.05). There was no significant difference between 1 and 2-week and the Sham group (P>0.05). (Table 2, figure2-3).
3.1.3. Expression of cytokine mRNA in cardiomyocytes of AMI mice
In myocardial tissue, the mRNA levels of TNF-α, IL-1, IL6, and BAFF in the AMI group were higher than those in the Sham group at 3 days, 5 days, 1 week, and 2 weeks (P < 0.05), and were highest at 3 days and 5 days subgroups (P < 0.05), however, the mRNA levels of TNF-α, IL-1, IL-6 and BAFF in AMI 3 days and 5 days subgroups were not statistically significant (P > 0.05). The level of TGF-β1 was higher than that of the 4 subgroups in the Sham group, and the highest level appeared at the 5-day subgroup (P<0.05). (Figure 4)
3.1.4. Levels of cytokines in peripheral blood of AMI mice
The concentrations of TNF-α, IL-1, IL6, and BAFF of peripheral blood in the AMI group were higher than those in the Sham group including 3 days, 5 days, 1 week, and 2 weeks subgroups (P < 0.05), and were highest at 3 days and 5 days subgroups (P < 0.05). TGF- 1 concentrations were higher than the Sham group in 4 subgroups, with the highest concentration in the 5 days subgroup (P < 0.05). The results were shown in figure 5.
3.1.5. Pathological changes of mice myocardium
In the AMI group, myocardial cells necrosis and inflammatory cells infiltration occurred at 3 days. The inflammatory cells infiltration was the most obvious at 5 days, the inflammatory cells infiltration decreased and a little fibrosis appeared at the 1-week subgroup. A few inflammatory cells and obvious fibrosis appeared at 2 weeks. Masson staining indicated that myocardial collagen deposition increased with time and reached its peak at 2 weeks. No obvious abnormalities were found in the myocardial pathology of Sham group mice. The pathological changes were shown in figure 6.
3.2. The effect of B cells on myocardial collagen metabolism after acute myocardial infarction in mice and its possible mechanism
3.2.1. Cytokine mRNA levels in myocardial tissue differences among the Sham group, WT group, and BKO group.
In 5 days subgroup of AMI, mRNA levels of TNF-α(2.33± 0.35VS3.79 ±0.58), IL-1 (2.87±0.35vs 5.32±0.37), IL-6 (3.35±0.28 vS6.58 ±0.48), and TGF-β1 (2.87±0.62 vS4.35 ±0.35) in BKO group decreased compared with WT group (P < 0.05), and were higher than those in Sham group (P < 0.05). ( Figure 7).
3.2.2. Peripheral blood cytokine concentration in myocardial tissue differences among Sham, WT, and BKO groups.
In 5 days subgroup of AMI, the concentrations of TNF-α, IL-1, IL-6, and TGF-β1 in peripheral blood of the BKO group decreased compared with the WT group (all P < 0.05), which were higher than the Sham group (P < 0.05), as shown in figure 8.
3.2.3. mRNA levels of collagen metabolism in the myocardium
The mRNA levels of COL1-A1 (3.60±0.65vs8.33±0.60), COL3-A1 (5.40±0.47vs2.46±0.30), MMP9 (3.84±0.71vs 8.19±0.99) of the AMI group, TIMP-1 (3.63±0.31vs6.23±0.56) of 2 weeks subgroup in BKO group were decreased compared with WT group (P < 0.05), but higher than those in Sham group (P < 0.05). (Figure 9).
3.2.4. Comparison of color doppler echocardiography in 2 weeks subgroup of Sham, WT and BKO group
The left ventricular end-diastolic diameter (LVEDd) left ventricular end-systolic diameter (LVESd) of the BKO group were smaller than those of the WT group (P<0.05), and the EF value was higher than that of the WT group (P<0.05). The LVEDd and LVESd were smaller than those of the Sham group, and the EF was lower than that of the Sham group. The difference was statistically significant (P<0.05). The results were represented in table 3 and figure 10.
3.2.5. Comparison of pathological findings in 2 weeks subgroup of Sham group, WT group, and BKO groups
Compared with the myocardial pathology of the mice in the WT group at 2 weeks, the myocardial fibrosis in the BKO group was reduced, and the collagen content decreased. The fibrosis in the BKO and WT groups was more obvious than that in the Sham group. The results are shown in Figure 11.