Evaluation of NRF2 Activator in Cardiac Cachexia

Abstract

EVALUATION OF NRF2 ACTIVATOR IN CARDIAC CACHEXIA BACKGROUND AND OBJECTIVE Cachexia is a predominant and significant pathological disorder related with perpetual cardiovascular breakdown. Its event predicts reduced survival, independent of pertinent factors, for example, age, ejection fraction, and heart’s physical capacity. Cardiac atrophy is a kind of catabolic remodelling brought about by a physiological reaction to chronically diminished heart workload or to complicated inflammatory disease. In the process of inducing heart failure induced cachexia, inflammatory cytokine-induced skeletal muscle changes play a significant role. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor which is expressed in most of the tissues and exhibit a major role in amplification of the antioxidant pathways associated with the enzymes present in myocardium. NRF2 plays a significant role in coordinating the cellular processes and protects the sensitive cellular components like proteins and DNA materials from oxidative insults. There have been many reports regarding therapy for cardiovascular diseases targeted through NRF2 receptor. The drugs which are already reported to be NRF2 activators have reported side-effects which couldn’t be neglected and some which provide a slow action. Therefore, there arises a need for a drug which activates NRF2 receptor and is safe as well as efficacious. Ezetimibe is reported to activate Nrf2-Keap1 pathway without causing cytotoxicity. Until now, Ezetimibe is revealed to promote the defensive effect of Nrf2 against lipid accumulation and having the potential in the prevention and treatment of inflammatory diseases. Despite this, no data is available with respect to effect of ezetimibe in cardiac cachexia. Therefore, the main objective of this investigation is to investigate the role of Ezetimibe, a NRF2 activator, in cardiac cachexia and to develop treatment strategy for cardiac cachexia. MATERIALS AND METHODS Balb/c mice of either sex at 6-8 week of age, weighing 25-35 gm were chosen for the study and maintained under well-controlled conditions of temperature (22 ± 2 ̊C), humidity (55 ± 5 %) and 12h/12h light-dark cycle. Standard laboratory rat chow and UV-filtered water was provided. The mice were injected intraperitoneally (i.p.) with (2 mg/kg) doxorubicin in 0.9% sodium chloride solution for alternate days for first week and then once a week for next 4 weeks. Controlled mice received equivalent amount of isotonic saline alone. Treatment with Ezetimibe was started from the 4th week. Ezetimibe (1.5 mg/kg) was dissolved in 0.5% methyl cellulose and given orally in doxorubicin induced cardiac cachexia animals. Animals were maintained with free access to conventional dietary feed and water ad libitum throughout experimental period. Parameters evaluated were body mass markers, inflammatory markers, markers of carbohydrate metabolism, marker of lipid metabolism, cardiac atropghy markers, skeletal muscle wasting marker. RESULTS Body mass marker: In the cardiac cachexia control animals, there was an obvious decrease in the body weight, food and water intake. Whereas, if we compare the cardiac cachexia control animals and ezetimibe treated animals, there is a significant improvement in the % body weight reduction. We noted that there was a noteworthy decrease in the carcass weight in the cardiac cachexia control animals while in ezetimibe treated animals, there was a significant improvement in the carcass weight. Markers of lipid metabolism: Doxorubicin-induced cardiac cachexia control animals showed a significant increase in the total cholesterol, LDL, triglyceride and VLDL level as compared to normal control animals. After the 4 weeks treatment with ezetimibe, there was a substantial decrease in the total cholesterol, LDL, triglyceride, HDL, VLDL level as compared to cardiac cachexia control animals. Further, the HDL levels are observed to decrease significantly in the cardiac cachexia control animals in comparison to control animals. There was an obvious decrease in weights of white and brown adipose tissues in the cardiac cachexia control animals. After the ezetimibe treatment, there was a substantial increase in the weights of white and brown adipose. Cardiac complication marker: A significant increase in the CK-MB and LDH levels in cardiac cachexia control animals when compared to normal control animals was observed. After the treatment with ezetimibe, a substantial decrease in CK-MB and LDH levels was observed as compared to cardiac cachexia control group. Cardiac atrophic markers: The cardiac atrophic index was significantly lower in cardiac cachexia control animals when compared to normal control animals. Ezetimibe treatment produced a significant increase in the cardiac atrophic index compared to cardiac cachexia control animals. The heart weight to body weight ratios (HW/BW), right ventricular weight to heart weight ratios (RV/HW) and left ventricular weight to heart weight ratios (LV/HW), which are the markers of cardiac atrophy, were observed to be significantly decreased in cardiac cachexia control animals when compared to normal control animals. Treatment with ezetimibe produced a significant increase in the HW/BW, RV/HW and LV/HW ratios compared to cardiac cachexia control group. Inflammatory marker: A substantial increase in the CRP levels in the cardiac cachexia control animals was observed. After the treatment with ezetimibe, there was a substantial decrease in the CRP. Markers of carbohydrate metabolism: There was a noteworthy increase in the serum glucose levels in the cardiac cachexia control animals. After the treatment with ezetimibe, there was a substantial decrease in the serum glucose levels relative to cardiac cachexia control animals. Skeletal muscle wasting marker: The weights of the skeletal muscles such as EDL, gastrocnemius, soleus, tibialis anterior, quadriceps muscles were significantly lower in cardiac cachexia control animals relative to normal control animals. Ezetimibe treatment produced a significant increase in the weights of EDL, gastrocnemius, soleus, tibialis anterior, quadriceps muscles when compared to cardiac cachexia control animals. CONCLUSIONS Our data suggest that NRF2 activator, Ezetimibe produces beneficial effect on cardiac cachexia in Doxorubicin induced cardiac cachexia model. Ezetimibe was successful to reduce the levels of inflammatory cytokines, ameliorate the effects on cardiac muscle wasting, lipid levels, fat tissues and skeletal muscles.