Interleukin-6 expression and its modulation by diacerein in a rat model of chronic stress induced cardiac dysfunction

Abstract

People with chronic stress have higher levels of pro-inflammatory cytokines, which enhance their susceptibility to cardiovascular diseases. Diacerein has ability to modulate pro-inflammatory cytokines such as IL-1β and IL-6; however, its efficacy in chronic stress associated cardiovascular diseases is not yet assessed. In this study, we standardized a rat model of chronic unpredictable stress (CUS) demonstrating cardiovascular dysfunctions and further assessed the effect of IL-6 modulator, diacerein, on cardiovascular functions in CUS exposed rats. The CUS procedure consisted of exposing male albino Wistar rats to random stressors, everyday for 8 weeks. The binding affinity of diacerein with IL-6 was ascertained using Docking tools viz AutoDock and SwissDock. Moreover, diacerein was administered (50 mg/kg/day x 20 days P.O) post CUS exposure to rats and the serum IL-6 levels and heart functions of CUS rats were determined by ELISA and ECG-HRV analysis, respectively. 8 weeks of CUS exposure resulted in two-fold increase in serum corticosterone and IL-6 levels in rats. The ECG and HRV analysis of CUS rats showed altered sinus rhythm, elevated heart rate, systolic blood pressure and sympathetic tone. Molecular docking studies revealed diacerein high binding affinity towards IL-6 receptor. The post-treatment of diacerein in CUS rats prevented these cardiovascular dysfunctions. Our findings thus suggests that IL-6 may have a prominent role in chronic stress induced cardiovascular dysfunctions and diacerein, could be used as a preventive measure for such conditions.

Keywords: Cardiac dysfunction; Chronic unpredictable stress; Diacerein; Interleukin-6; Pro-inflammatory cytokines.

Figure 1

A schematic representation of the experimental design and the timeline of experiments. (A) Initially rats were randomly assigned to two groups Control and CUS wherein control rats were gently handled during the course of study and CUS group rats were exposed to 8 weeks of chronic unpredictable stress (CUS) followed by standardisation. CUS group was further randomly divided in two groups: Vehicle (CUS-VEH) and CUS-DCN, wherein CUS-DCN group received diacerein (50 mg/kg in 0.2% CMC) for 20 days and Vehicle group received vehicle alone for 20 days. Post treatment both groups were assessed for physiological and biochemical changes. (B) The CUS paradigm consisted of exposing rats randomly to following stressors daily for 8 weeks: restraint – rats were restrained in a restrainer for 1 hour; shaking - rats were shaked in a shaker for 1 hour; social defeat – the rats were placed in a cage along with an aggressive rat, and after being defeated, they were returned to their former cages; hot air stream – rats were subjected to hot air stream from a hair dryer for 10 min; overnight illumination – rats were constantly exposed to light during day as well as night time; inverted light cycle – the regular lighting environment in the animal room was reversed (off during the day and on at night); tilted cage – the rat cage was inclined up to 45° angle for 1 hour.

Figure 2

Standardisation of CUS paradigm in rats. (A) The body weight of control and CUS group were measured every week, before the start of stress protocol till the end. The CUS group animals gained less weight compared to control group. (B) Body weight assessment revealed significantly less weight variation (%) in CUS rats depicting weight loss as well as less weight gain in rats exposed to 8 weeks of CUS. (C) In force swimming test the immobility time was found significantly increased in CUS rats compared to control. (D) Elevated plus maze test revealed increased time spent in closed arms by CUS rats compared to control group. (E) Novelty suppressed feeding test revealed increased latency to the first bite by CUS rats, revealing depressive behaviour. (F) Serum corticosterone level was significantly elevated in CUS rats after 8-week of stress paradigm. All parameters were compared between the CUS and control groups using unpaired t-test (∗P<0.05, ∗∗ P<0.01, ∗∗∗ P<0.001, ∗∗∗∗ P<0.0001).

Figure 3

Effect of chronic unpredictable stress on cardiac function in CUS rats. (A–B) represents the average ECG pattern of a sample control and CUS rat, respectively. CUS rat has significantly altered R amplitude, T amplitude with elevated ST segment compared to control. (C) represents the effect of CUS on various ECG parameters. ECG analysis revealed significantly elevated heart rate (HR), R Amplitude T Amplitude along with decreased RR interval, P duration and QRS interval in CUS rats. (D) represents the effect of chronic stress on various HRV indices. HRV analysis by LabChart revealed that chronic stress resulted in reduced HF, RMSSD, SD1, indicating dominance of SNS and diminished vagal tone. All parameters were compared between the CUS and control groups using unpaired t-test (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Figure 4

Docking studies of diacerein with IL-6 (PDB:1N26). (A) The output of AutoDock 3D diagram shows the binding site residues of IL-6 protein with the diacerein, with binding affinity of -8.44 kcal/mol. The residues in the binding site are shown in green color. Diacerein is shown in gray color. (B) The 2D structure exhibits different types of interactions formed between IL-6R and diacerein. The dark green and purple dotted lines indicate H-bond and Pi-Alkyl interactions between IL-6R and diacerein, respectively. (C) The interaction result of IL-6R with the diacerein and the obtained ΔG value was -7.22 kcal/mol.

Figure 5

Effect of diacerein on IL-6 expression in CUS rats. Serum analysis revealed significant rise in IL-6 levels in CUS rats, which was significantly reduced in rats treated with diacerein. Values are presented as Mean ± SD; each group contains 6 animals. All parameters were compared to CUS group using one-way ANOVA test (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Figure 6

Effect of diacerein treatment on ECG and HRV parameters of CUS rats. Box-cum-whisker plots showing quantitative variations in relative signal integrals of various important components of ECG and HRV. In the box plot, the boxes denote interquartile ranges, the horizontal line inside the box denotes the median, and the bottom and top boundaries are 25^th and 75^th percentiles, respectively. Lower and upper whiskers are 5^th and 95^th percentiles, respectively.

Figure 7

PCA, PLS-DA and VIP score of ECG and HRV. (A–B) represents the PCA and PLS-DA plot of ECG parameters involving all groups, respectively, with statistical values in the form of regression (R2 0.85). (D–E) represents the PCA and PLS-DA plot of HRV parameters involving all groups, respectively, with statistical values in the form of regression (R2 0.58). Circles indicate the 95% confidence interval for each class. The LOOCV method is used in cross-validation, using accuracy as a performance measure in the MetaboAnalyst web server. (C&F) represents the VIP scores of ECG and HRV in decreasing order of their respective VIP score values. G1, G2 and G3 indicate control, CUS-VEH and CUS-DCN groups, respectively.

Figure 8

Effect of diacerein treatment on CUS induced rise in systolic blood pressure. The CUS paradigm resulted in an increase in systolic blood pressure. This rise in systolic BP was prevented in diacerein-treated CUS rats. Values are presented as Mean ± SD; each group contains 6 animals. All parameters were compared to CUS group using one-way ANOVA test (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).