Role And Mechanisms Of Celastrol In Treatment Of Obesity In Rats

Background Since 1980,the global prevalence of obesity has increased about 2 times.There are about 1.9 billion people suffering from overweight and obesity around the world.With the development of the society and economy,obesity and its related diseases such as diabetes and cardiovascular diseases are getting great risk that threaten the health of the Chinese.Health problems caused by obesity have contributed to an unbearable burden on individuals,society,medical care,and economy,which becomes one of the most severe public health issues in the 21 st century in the world.It is so imminent to discover and develop a safe and effective treatment of obesity.The current treatments for obesity mainly include diet restriction,exercise,surgery,and drugs.Among them,surgical operations mostly reduce the stomach volume and stimulate the vagus nerve reflex,resulting in simulated satiety to reduce food intake.Besides surgery,drug therapy is also an option.The principle of treatment is stimulating a reduction in energy intake and/or an increase in energy expenditure,thereby negatively regulating the energy balance.However,most of the patients tend to lose weight transiently but not maintain for a long time.It is mainly because the feeding center that manages appetite and satiety in the hypothalamus and the reward system that manages pleasure behavior are connected in both of the structure and function,making eating often synergetically occur with the happiness.Moreover,such the synergy is closely related to emotion and cognition.And that makes the patient's insistence on obesity often come from an instinctive,physiological adherence,which makes it very difficult for treatment.The gastrointestinal tract plays an important role in exchanging substances inside and outside the body,where microbiomes promote carbohydrate and lipid metabolism and maintain energy balance.Mounting evidence shows that decline in the microbial diversity is associated with the occurrence of obesity and other metabolic diseases.On the other hand,the transplantation assay reveals that microbiomes from normal-weight donors have the protective effect against the obesity caused by high-energy diets.It makes obesity treatment by reversing and improving the gastrointestinal environment homeostasis through diet or drugs become true.In recent years,various natural plant-derived biomolecules including resveratrol,quercetin,andrographolide and berberine have been proven to be beneficial in controlling metabolic diseases.Celastrol,a purified extract of Chinese medicine Tripterygium wilfordii,has a strong anti-oxidative effect and is widely used in anticancer angiogenesis.Previous studies have reported that celastrol can inhibit the appetite of obese mice,stimulate energy consumption by activating energy metabolism pathways of the fat and muscle and prevent from the impact of high-fat diet,introducing celastrol as a potentially effective drug for obesity treatment into the vision of public.However,there is still great controversy regarding the dose,kinds of administration,and the onset interval of the effects of celastrol on obesity.In addition,it still remains poorly understood whether celastrol has an effect on the gut microbiome.Therefore,this study used a high-fat diet-induced obese rat to identify the body weight,food intake,and energy metabolism,combined with high-throughput sequencing and gene editing techniques,to verify the effects of celastrol and to explore the potential roles played by the gut microbiome.MethodsFirstly,we used male ICR mouse as the model to test the acute toxicity of celastrol.All mice were intragastrically administered celastrol at a dose ranging from 0 to 62.5 mg/kg b.w.at one time.We observed the general status of mice for 5 consecutive days by body weight,feeding and spirit.The safe dosage range of celastrol was determined by equivalent dosage conversion from mouse to rat.Subsequently,we established obese SD rats by feeding 60% high-fat diet from 9-weekold to 20-week-old.Doses of 0 ?g/kg,15 ?g/kg,50 ?g/kg,100 ?g/kg,150 ?g/kg,200 ?g/kg,250 ?g/kg and 500?g/kg of celastrol were adiministered to rats for 21 consecutive days by intraperitoneal injection and intragastric administration respectively.By recording the body weight and food intake of the rats,measuring blood serum biochemical indices,staining H&E sections of abdominal white fat and liver,and evaluating oral glucose tolerance and insulin tolerance test,we identified effective dose of celastrol for obesity treatment.Afterwards,we collected the feces from the solvent control and celastrol treatment group at different time points,extracted the genomic DNA of microbiomes under aseptic conditions,performed 16 S r RNA amplicon sequencing on the V3 region,and analyzed differences in diversity and relative abundance of gut microbiota between the two groups by the Qiime and R:Phyloseq softwares.On this basis,we added neomycin,penicillin,vancomycin,and metronidazole into drinking water to interfere with the gut microbiota and observed its influence on the treatment of obesity with celastrol.By collecting hypothalamus tissues from control and treated rats,we extracted RNA and performed transcriptome sequencing.We used SAMtools,HTSeq and R:DESeq2 software to analyze differentially expressed genes.Pathway enrichment analysis was conducted by IPA software.Obtaining essential gene knockout SD rats by Crispr-Cas9,we treated them with celastrol,observed phenotypes of rats,and explored the roles of that gene played in the homeostatic control of energy balance.Finally,we combined the TSE metabolic monitoring system,which detected changes in food intake and energy metabolism in rats after treatment with celastrol,and gene expressions of oxidative phosphorylation pathway in brown and white adipose tissues to study the functions and mechanisms of celastrol exerted on regulating the energy balance and thus curing obesity.ResultsWe found that there was no lethal acute toxicity of celastrol in male ICR mice within a dose range from 0 to 62.5 mg/kg b.w.According to the equivalent dosage conversion of drugs between different species,the dose was about half that of mice in rats.Thus,celastrol could be safely used in rats at a dose of less than 30 mg/kg b.w.Rats were treated with celastrol for 21 consecutive days.During the treatment,rats in the solvent control group remained stable in body weight,and there were no abnormalities in general conditions such as feeding,spirit,and activity.As to celastrol treatment group of 500 ?g/kg b.w.,the body weight of rats gradually decreased.By the 21 day,the body weight of rats decreased by an average of 25.7 g.There was a statistical difference in the percentage of decrease in body weight between the two groups.H&E staining sections showed that white adipose tissue was normal in both groups,and there was no significant difference in lipid droplet size.But rats in the solvent control group had more lipid droplets in the liver than that in the treatment group.There were no significant differences in glucose,triglyceride,total cholesterol,high-density lipoprotein,and low-density lipoprotein levels between the two groups.The oral glucose tolerance of the two groups remained impaired,and the insulin tolerance of the rats treated with celastrol was slightly improved.16s r RNA analysis of gut microbiota showed that there was a significant difference in ?-diversity between the two groups,and the ?-diversity of treatment group was significantly increased.Relative abundance of Bacteroidetes was up-regulated, whereas that of Firmicutes was decreased.In addition,the relative abundance of gut microbiomes changed with the process of treatment of celastrol,and this change stabilized after two weeks of treatment.The ratio of Bacteroides to Firmicutes showed a simultaneously changing tendency.After interfering with antibiotics,the ?-diversity of microbiota in the interference group was lower than that of the single celastrol treatment group,and the speed of weight loss of interference group was slowed down.By the end of the experiment,there was a large difference in the percentage of decrease in body weight between the two groups.Transcriptome sequencing results showed that there were 153 up-regulated genes and 13 down-regulated genes in the hypothalamus of rats treated with celastrol.Pathways including G protein-coupled receptor signaling pathway,dopamine receptor signaling pathway,and leptin signaling pathway were significantly enriched.Leptin activates downstream POMC and NPY/Ag RP neurons to suppress appetite and promote energy expenditure by binding receptors in the hypothalamus,and it plays an important role in regulating the energy balance,so we then focused on the function of leptin signaling pathways.The level of serum leptin in the treatment group showed a decreasing trend throughout the experimental period,but no significant difference was observed between the two groups.Stat3 is a direct target in leptin signaling pathway,the level of Stat3 between two groups did not differ significantly.And intensity of phosphorylated Stat3 in the celastrol treatment group was significantly increased,indicating the activation of leptin signaling pathway in this group.In heterozygous Leptin knockout rats,celastrol did not decrease the body weight at a dose of 500 ?g/kg b.w.until it was increased to 1000 ?g/kg b.w.There was a significant difference in percentage of decrease in body weight between the control and treatment groups.However,no weight loss was observed at a dose of 1000 ?g/kg b.w.of celastrol in homozygous Leptin knockout rat.The results of energy metabolism showed that,after treatment with celastrol for one week,the respiratory exchange rate and activity of the rats between two groups were not statistically different,while energy expenditure of the rats in the treatment group was significantly higher than that of the control group during both day and night.The expressions of fatty acid metabolism pathway genes,including Adipoq,Fasn,Fabp4 and Lpl,in the brown adipose tissue of the treatment group were up-regulated,and the difference was statistically significant.No such change was observed in the white adipose tissue.We further compared the energy metabolism of wild-type and homozygous Leptin knockout rats treated with the same dose of celastrol and found that the energy expenditure of Leptin knockout rats was significantly lower than that of wild-type ones.And there were no differences in respiratory exchange rate and activity level.Conclusions The present study found that:(1)Celastrol did not show acute lethal toxicity at a dose of less than 62.5 mg/kg b.w.in mouse,and gavage administration was safer than intraperitoneal injection.(2)Celastrol decreased body weight,improved insulin sensitivity and enhanced energy expenditure at a minimum dosage of 500 ?g/kg b.w.,but there was no effect on food intake and blood serum biochemical indices.(3)Celastrol ameliorated the diversity of gut microbiomes and increased the ratio of relative abundance of Bacteroidertes to Firmicutes.(4)Leptin signaling pathway was involved in the process of celastrol enhancing the energy expenditure which could be mediated by gut microbiomes.This study initially evaluated the toxicity of celastrol and systematically examined the therapeutic effect and mechanism of celastrol on obesity in rats,providing an exact scientific reference and concrete technical support for the potential clinical application of celastrol.