Running title: Heart Failure Treatment
- The level of TNF-α decreased after treatment with IL-10 and golimumab.
- The level of IL-10 was increased by IL-10 combined with golimumab.
- IL-10 and golimumab have a synergistic effect on HF in rats.
Objectives We attempted to assess the therapeutic effect of exogenous interleukin-10 (IL-10) in combination with golimumab in treating heart failure (HF) in rats, and provide an experimental basis for clinical application.
Methods HF animal model was induced in Sprague-Dawley (SD) rats by intraperitoneally injection with adriamycin, and rats in control group were treated with 0.9% normal saline. The HF rats were randomly divided into 4 groups: group A (HF group, n = 10), group B (IL-10 group, n = 10), group C (golimumab group, n = 10), and group D (IL-10 and golimumab group, n = 10). The normal rats were defined as controls in group E (n=10). Three months later, myocardial functional indexes and hemodynamic indexes of rats in all groups were measured. The levels of TNF-α and IL-10 in serum were detected by enzyme linked immunosorbent assay (ELISA).
Results The HF rat model was successfully constructed. After the treatment, compared with group A, the hemodynamic indexes in groups B-D were distinctly different (P < 0.05), and these in group D were most notably different (P < 0.01). Compared with group A, the level of TNF-α in group D was obviously decreased (27.38±2.45 pg/ml vs. 45.35±3.26 pg/ml, P < 0.05), whereas IL-10 significantly rose (643.66±78.43 pg/ml vs. 275.58±56.62 pg/ml, P < 0.01).
Heart failure (HF) is considered to be a condition in which the heart can hardly pump sufficient blood to meet the need of the body . It was demonstrated that HF is closely related to several diseases such as hypertension, ischemic heart disease, diabetes, obesity and dyslipidaemia . HF has already been a major cause of morbidity and mortality worldwide [3, 4]. It was reported that more than23 million people in the world are suffering from HF . Mortality caused by HF is up to 30%-40% within one year, and rise to a rate of 50%-75% within 5 years [5, 6]. So, HF has been a health concern all over the world.
Previous studies suggested that the imbalance of anti-inflammatory and pro-inflammatory cytokines played a key role in the progression of HF . Recent studies have highlighted the effects of some cytokines on immune-mediated myocyte injury, postischemic myocardial inflammation, and heart function. Levels of anti-inflammatory cytokines such as interleukin-10 (IL-10) are reduced in HF development [8-11]. Evidences demonstrated IL-10 controlled inflammation by inhibiting pro-inflammatory cytokine synthesis and inducted antiapoptotic processes . IL-10 can promote healing of ventricular damage to avoid ventricular remodeling [13, 14]. IL-10 can enhance humoral immunity by inhibiting cell-mediated immunity to resist HF. Thus, IL-10 is considered to be a candidate for the management of HF, . Serum level of pro-inflammatory cytokines of tumor necrosis factor α (TNF-α) are found to be elevated in experimental models of HF . Studies declared that an increase of TNF-α concentration resulted in necrosis and apoptosis of myocardial cells, finally led to ventricular remodeling by the progressive thinning and expansion of ventricular wall . TNF-α was reported to lower the myocardial systolic function directly [13, 14]. Furthermore, evidences uncovered the relationship of TNF-α and progression of HF: elevated level of TNF-α contributed to the development and progression of HF by activation of matrix metalloproteinases, and promotion of myocardial hypertrophy and contractile dysfunction .
Therefore, the TNF-α inhibitors, as medicines for HF treatment, had been becoming the current hot topics in HF studies. Nowadays, etanercept, a recombinational TNF-α inhibitor, had been synthesized already, which inhibit the pro-inflammatory of TNF-α due to the interdicted effect for the conjugation of TNF-α and cell membrane receptor . Golimumab, another TNF-α inhibitor, is a monoclonal antibody specific for human TNF-α . It can inhibit the bioactivity of TNF-α via binding to TNF-α with high affinity and specificity to prevent TNF-α from combination with TNF receptor . In addition, IL-10 has been shown to play a pivotal role in the mucosal immune system by inhibition of pro-inflammatory cytokine synthesis such as TNF-α [20, 21]. However, there are few studies reporting the effect of exogenous IL-10 combined with golimumab forthe HF treatment.
In the present study, we used HF rats model, which was induced by intraperitoneal injection with adriamycin (ADR), to explore the effect of exogenous IL-10 combined with golimumab in the treatment for HF. To analyze the therapeutic effects, IL-10 or/and golimumab were intraperitoneally injected into the HF rats and the effects of the treatments were tested by measuring heart functional indexes and level of inflammatory cytokines. In addition, the possible mechanisms of this effect were investigated.
MATERIALS AND METHODS
Fifty 10-week-old Sprague-Dawley (SD) rats, including 25 females and 25 males, were provided by Laboratory animal center, southern medical university Southern Medical University. Forty rats, with 20 females and 20 males, were chosen at random. Rats with HF were induced by intraperitoneal injection with 4 mg/kg adriamycin (ADR) once a week for 6 times. Control rats were given the same volume of normal saline.
Experimental design and drug administration
After 6 weeks, the model rats were randomly divided into 4 experimental groups: HF group (group A, n=10), IL-10 group (group B, n=10), golimumab group (group C, n=10), and IL-10 and golimumab group (group D, n=10), with 5 females and 5 males in each group. The normal rats were considered as controls in group E (n=10). Rats in group B was given an intraperitoneally injection of 50 pg/kg IL-10 each week. Exactly, 1 mg/kg golimumab (Centocor Ortho Biotech Inc) was administered intraperitoneally into the rats in group C each month. Rats in group D was intraperitoneally injected with 50 pg/kg IL-10 each week and 1 mg/kg golimumab each month. At the same time, rats in groups A and E received an intraperitoneal injection of 1ml normal saline. Rats in each group were treated for 3 times in total. During the test period, rats were fed on food and clean water freely.
Three months after administration, echocardiographic indicators were obtained using a color doppler ultrasonic cardiograph (Vingmed CFM-725). Rats were anaesthetized with 5 ml/kg urethane solution at a concentration of 20% by intraperitoneal injection, and left ventricular long-axis and M-mode curve at the level of the left papillary muscles were measured. Finally, the left ventricle end-diastolic diameter (LVEDD), the left ventricle end-systolic diameter (LVESD), left ventricular posterior wall depth (LVPWD) were measured, and the left ventricle fractional shortening (LVFS) can be calculated by the factors above, the formula is as follows:
ã€€LVFS = [( LVEDDï¼LVESD)/LVEDD]×100%.
Measurements of haemodynamic parameters
After anaesthetization, rats were fixed followed bytracheal intubationwith artificial ventilation. Super-miniature cardiac catheter was connected to multipurposepolygraph (Nikon 4) through the right common carotid artery and ascending aorta. Systolic pressure (ASP), diastolic pressure (ADP) and heart rate (HR) was recorded. As a follow-up, the cardiac catheter were inserted into the left ventricle, the left ventricle systolic pressure (LVSP), the left ventricular end-diastolic pressure (LVEDP) and the maximum rate of the left ventricular pressure rise (+dp/dtmax) and decline (-dp/dtmax) were measured. Then, allratsweresacrificed to weight the body. Their hearts were weighted after washing by cold normal saline and drying by filter paper. Finally, Heart weight/Body weight (HW/BW, mg/g) was calculated based on HW and BW.
Determination of the level of TNF-α and IL-10 in serum
After hemodynamic evaluation, 2 ml blood samples were drawn from the catheter positioned in the left common carotid artery, and collected into a 2 ml tube. After 30 min, the blood samples were centrifuged at 3000 r/min for 10 min. The supernatant were collected into Eppendorf (EP) tube. The concentration of TNF-α and IL-10 in serum were detected by using an ELISA kit (RayBiotech. Inc), and experimental procedures were conducted according to themanufacturer’sinstructions stringently.
The data were represented as mean ± standard deviation (SD). Comparisons between groups were analyzed byone-way ANOVA and q-test (Student-Newman-Kueulsmethod). All statistical analyses were performed by using SPSS 12.0 software (SPSS, Chicago, IL). P < 0.05 was considered to be statistically significant.
When the model establishment was completed, all of the rats suffered from HF, showing symptoms including dyspnea, edema, feed difficulty and oliguria. HF symptoms of the rats in groups B-D were alleviated after the treatment with IL-10, golimumab, and IL-10 and golimumab respectively. Obviously, symptoms of the rats in group D were most significantly alleviated. There was no rat died during the experimentation.
As shown in table 1, the BW and HW of the rats in groups A-D decreased significantly in contrast to those in group E (P < 0.05), whereas the HW/BW increased (P < 0.05). The BW, HW and HW/BW of the rats in groups B and C did not change significantly when compared with group A (P > 0.05). The BW and HW of the rats in group D were markedly increased compared with group A (422.85±34.45 mg vs. 315.45±20.56 mg; 1355.29±112.23 mg vs. 1154.58±135.48 mg, P < 0.05), however, HW/BW decreased obviously (3.21±0.10 mg vs. 3.66±0.18 mg, P < 0.05).
The results of echocardiography
Table 2 presents the results of echocardiography in the rats. Compared with E group, LVEDD and LVESD of the rats in groups A-D increased significantly (P < 0.05), to the contrary, LVPWD and LVFS decreased (P < 0.05). LVEDD and LVESD in group D decreased obviously compared with group A (7.45±0.45 mm vs. 9.45±0.26 mm; 3.69±0.23 mm vs. 6.58±0.22 mm, P < 0.01), and lower than these of groups B and C. While LVFS(50.47±6.48 %) in group D was higher than group A (30.37±4.35 %, P < 0.01) as well as groups B and C. LVPWD in groups B-D didnotchangesignificantly (P> 0.05).
The hemodynamic indexes changes
Hemodynamic parameters of the rats in group A had a difference in statistical significance when compared with group E (table 3). After treated with IL-10 and golimumab, ASP and ADP in group D was significantly increased when compared with group A (155.89±23.45 mmHg vs. 108.35±35.58 mmHg; 114.26±13.65 mmHg vs. 114.26±13.65 mmHg, P < 0.01), and it was higher than groups B and C. LVSP in group Dmost markedly increased compared with group A (125.58±28.87 mmHg vs. 85.25±15.34 mmHg, P < 0.01) as well as groups B and C. LVEDP in group D (5.23±0.68 mmHg) was most significantly decreased contrasted with group A (12.45±1.21 mmHg, P < 0.01), and also lower than that of groups B and C. The decreased ±dp/dtmax in group D was significantly improved by treatment with IL-10 and golimumab (3988.35±165.45 mmHg/s) compared with group A (2989.68±109.35 mmHg/s, P < 0.01), while those in group B and C didn’t show significant difference.
Level of serum TNF-α and IL-10
Compared with group E, the level of serum TNF-α in groups A-D rose obviously (P < 0.05, shown in table 4). However, after treatment, the level of serum TNF-α in groups B and C compared with group A did not change distinctly, whereas, decreased obviously in group D (27.38±2.45 pg/ml vs. 45.35±3.26 pg/ml, P < 0.05). Conversely, compared with group E, the level of serum IL-10 in groups A-D declined obviously. The level of serum IL-10 in groups B, C and D was higher than group A, moreover, the level of serum IL-10 most significantly increased in group D when compared with group A (643.66±78.43 pg/ml vs 275.58±56.62 pg/ml, P < 0.01).
Heart failure (HF), as a complex clinical syndrome, is the ultimate end-result of various cardiovascular diseases and the main cause of death . HF prevention and treatment have drawn more and more attention inthemedicalfield. It has been reported that TNF-α played a central role in the progression of HF . Studies indicated that golimumab, as a monoclonal antibody, had effective on inflammation based on inhibition on pro-inflammatory cytokine of TNF-α [18, 19], moreover, the effect of TNF-α in many tissue es is modulated by IL-10 . In the present study, we used HF rats model to determine the effect of exogenous IL-10 combined with golimumab in the treatment for HF. The therapeutic effect of IL-10 combined with golimumab for HF in rats was evaluated according to heart function (changes of heart weight, echocardiographic and hemodynamic indicators) and the variation in the level of serum IL-10 and TNF-α.
To the best our knowledge, heart hypertrophy ultimately leads to HF , and the increase of HW/BW represents a tendency of heart hypertrophy . In the present study, no obvious changes of HW/BW were observed in groups B (IL-10 group) and C (golimumab group) compared with group A (HF group). But HW/BW in group D (IL-10 and golimumab group) was markedly decreased obviously when compared with group A (HF group). These results indicate that treatment with either IL-10 or golimumab is less effective than the combination of IL-10 and golimumab for relieving heart hypertrophy. Studies indicated that treatment targeted the pathological disease such as heart hypertrophy was effective for HF .Therefore, our results suggest IL-10 combined with golimumab may have significant effects on prevention of HF.
In addition, previous studies suggested that elevated LVEDD and LVESD and declined LVFS led to ventricular remodeling and culminated in progressive HF . Based on the echocardiographic data in the present study, we found that LVEDD and LVESD decreased markedly, and LVFS increased in groups B-D compared with group A. Particularly, the most remarkable variation was observed in group D. these results suggest that IL-10 combined with golimumab are more effective inthe treatment of HF by relieving ventricular remodeling.
Moreover, changes of hemodynamic parameters are regarded as an important signal of myocardial function which results in HF development . In the present study, the hemodynamic measurements were improved in group D compared with groups A by increasing ASP, ADP, LVSP, LVEDP, and ±dp/dtmax and decreasing LVEDP. Our observations were consistent with the previous findings showing that LVSP, LVEDP, and ±dp/dtmax were increaded and LVEDP was decreased after completing the study of the HF rats. Furthermore, the previous studies declared that the deterioration of myocardial function in HF rats was more significantly alleviated based on the improvement of hemodynamic measurements after treatment [26, 27]. Notably, in our study, hemodynamic parameters in group D were improved compared with groups B and C. Therefore, the results of this study suggest that IL-10 and golimumab may have synergistic effects on improvement of myocardial function to relieve HF symptoms.
Furthermore, in the present study, the level of IL-10 and TNF-α were detected. The level of serum IL-10 was elevated in groups B-D compared to group A. Similarly, studies observed that an increase in IL-10 content was showed after effective treatment and suggested that improvement in cardiac function was connected with the elevated level of IL-10 [28-30]. Moreover, level of serum IL-10 in group D was higher than groups B and C, this suggest that the combination of IL-10 and golimumab can most significantly induce the elevation in the level of serum IL-10. Meantime, the results of our study showed that the level of serum TNF-α in the rats of HF was significantly declined in group D compared with group A, while we noted that the treatment with IL-10 or golimumab only affected little the concentration of serum TNF-α in groups B and C. This led us to hypothesize that golimumab is less effective in inhibition of the expression of TNF-α, but more effective when combined with IL-10. As previous studies declared, TNF-α plays a central role in initiating and sustaining the inflammatory response  and is a mediator of cardiac pathology acting through inflammatory pathways and activation of myocardial apoptosis [32, 33]. On the other hand, IL-10 inhibits cytokine-induced oxidative stress along with affecting the expression of inflammatory cytokines . It is suggested that the imbalance between the IL-10 and TNF-α in the heart may result in an inflammatory response, which plays a pathogenic role in the development and progression of HF . The changes of the serum level of IL-10 and TNF-α in our study suggest that IL-10 and golimumab may modulate the inflammatory processes by promoting the expressing of IL-10 and suppressing the expression of TNF-α. Therefore, The combination of IL-10 and golimumab may be more effective than IL-10 or golimumab in the treatment of HF via inflammatory pathways.
In conclusion, exogenous IL-10 and golimumab had synergistic effects on HF in rats based on the improvement in myocardial function and prevention of heart hypertrophy and ventricular remodeling through inflammatory pathways. Furthermore, the synergistic effect of IL-10 and golimumab is superior to the effect of treatment with IL-10 or golimumab only. Our results suggest that the combination of IL-10 with golimumab may be a promising treatment for HF patients. Further study is warranted to verify the efficiency of IL-10 combined with golimumab in human clinical trials.