Celastrol Alleviates Gamma Irradiation-Induced Damage by Modulating Diverse Inflammatory Mediators
Authors: Arunachalam Chinnathambi
Hong Wang
Kwang Seok Ahn
Feng Ru Tang
Sulaiman Ali Alharbi
Frank Arfuso
Omar Hm Shair
Gautam Sethi
Journal: International Journal of Molecular Sciences (2020)
Discipline: CHEMISTRY
Keywords: NF-κB; ROS; celastrol; gamma radiation; in vitro
Affiliations: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117600, Singapore., Singapore Nuclear Research and Safety Initiative, National University of Singapore, Singapore 138602, Singapore., College of Korean Medicine, Kyung Hee University, #47, Kyungheedae-gil, Dongdaemoon-gu, Seoul 130-701, Korea., Department of Botany and Microbiology, College of Science, King Saud University, Riyadh-11451, Saudi Arabia., Stem Cell and Cancer Biology Laboratory, School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, WA 6009, Australia.

Abstract:  The present study aimed to explore the possible radioprotective effects of celastrol and relevant molecular mechanisms in an in vitro cell and in vivo mouse models exposed to gamma radiation. Human keratinocytes (HaCaT) and foreskin fibroblast (BJ) cells were exposed to gamma radiation of 20Gy, followed by treatment with celastrol for 24 h. Cell viability, reactive oxygen species (ROS), nitric oxide (NO) and glutathione (GSH) production, lipid peroxidation, DNA damage, inflammatory cytokine levels, and NF-κB pathway activation were examined. The survival rate, levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in blood, and p65 and phospho-p65 expression were also evaluated in mice after exposure to gamma radiation and celastrol treatment. The gamma irradiation of HaCaT cells induced decreased cell viability, but treatment with celastrol significantly blocked this cytotoxicity. Gamma irradiation also increased free radical production (e.g., ROS and NO), decreased the level of GSH, and enhanced oxidative DNA damage and lipid peroxidation in cells, which were effectively reversed by celastrol treatment. Moreover, inflammatory responses induced by gamma irradiation, as demonstrated by increased levels of IL-6, TNF-α, and IL-1β, were also blocked by celastrol. The increased activity of NF-κB DNA binding following gamma radiation was significantly attenuated after celastrol treatment. In the irradiated mice, treatment with celastrol significantly improved overall survival rate, reduced the excessive inflammatory responses, and decreased NF-κB activity. As a NF-κB pathway blocker and antioxidant, celastrol may represent a promising pharmacological agent with protective effects against gamma irradiation-induced injury. 

Retracted: No

PubPeer comments:
 Here, the authors expose skin keratinocytes and foreskin fibroblasts to 20 Gy gamma radiation at a dose rate of 2.6 Gy min. The cells appear to be so radioresistant - more than 75% percent cells survived at 24 hours after exposure to 20 Gy. this is rather strange. Any radiation biologists, please justify this. Mice were exposed to 6.4 Gy and followed until 30 days...only 4 mice per group were used. Very difficult to justify the results based on the doses used, end points studied and number of mice used in the experiments. , 
 #1 unfortunately, the authors ignored this question. According to this paper in 1972 : LD50(30)s and Daily Death Distributions of Whole or Partial Body Irradiated Mice F. Sato, S. Tsuchihashi, W. Nakamura, H. Eto Journal of Radiation Research, Volume 13, Issue 2, June 1972, Pages 100u2013108, and subsequent many other papers have clearly demonstrated that LD50 is lower than the dose the authors have used here. For 4 Gy of whole body irradiation, most mice will die within 30 days! Therefore, i sincerely doubt that all 4 mice survived 6.4 Gy of gamma radiation.. Authors write that Gamma-irradiation with 6.4 Gy induced 80% animal death at 30 days. Therefore, i assume that they had very few mice for analysis at 30 days..If they have used only 4 mice, this is not statistically relevant at all. , 
 Thanks for your comments. In vitro cultured cells are more radioresistant than in vivo animals. We had done preliminary tests on the effects of gamma irradiation with different doses and post-irradiation times and decided to employ 20 Gy. Cell death increased with the prolonged post irradiation time and higher doses of gamma irradiation exposure. ![file] Regarding animal results, we did preliminary tests. Please refer to the figures below. Regarding LD50 30, we started from 4Gy gamma irradiation. However, it did not cause any animal death in 30 days. When the dose increased, 5.33Gy caused two animal death in our female Balb c mice. 7.68Gy caused all animal death. Based on our observation, we finally selected 6.4Gy for the drug treatment. We also tested different concentrations of drugs as well as pre- and post-drug treatment, which have not been submitted for publication. Each group contained 10 mice, except drug treatment group without irradiation contained 5 mice in the effort to minimize animal numbers. Please note that LD50 30 may vary significantly due to SPF or non-SPF facility, animal health condition etc Hope these can clarify your doubts. ![file] , 
 #3 I am sorry to note that you have not submitted the data from 10 animals instead showed only from 4 animals. What is the reason for this. Unfortunately, if the data are not shown submitted published, it is difficult to convince readers, i believe.  When the dose increased, 5.33Gy caused two animal death in our female Balb c mice. - apparently, the data you have shown in the paper is only from 2 mice? How significant that can be? ,