The Mechanism of Regulating Leptin Resistance in Obesity and the In fluence of Adjusting Methylation of OB-R,POMC Gene Promoter of Wendan Decoction (温胆汤)

YANG Hai-yan (杨海燕), YU Song-ren (喻松仁), WANG Ping (王 萍),SHU Qing (舒 晴), CHENG Shao-min (程绍民)

1. Science and Technology College, Jiangxi University of traditional Chinese Medicine, Nanchang 330000, China

2. College of Traditional Chinese Medicine, Jiangxi University of traditional Chinese Medicine, Nanchang 330000, China

3. Graduate student of Jiangxi University of traditional Chinese Medicine, Nanchang 330000, China

ABSTRACT Objective:To explore the mechanism of Wendan Decoction (温胆汤) regulating leptin resistance in obesity by analyzing the level of leptin in peripheral blood, the expression of leptin receptor and signal pathway molecule POMC in hypothalamus . To analyze the methylation status of the gene promoters of leptin receptor and POMC in obese rats fed with high fat diet, and the in fluence of adjusting methylation of OB-R, POMC gene promoter about Wendan decoction.Methods:Model rats were randomly divided into model control group and drug observation group, additional normal rats with 8 rats in each group. The models were established by high fat diet. The observation group was treated with Wendan Decoction (温胆汤) and the other two groups were treated with sterilized drinking water once a day for 6 weeks. Body weight was recorded every week during the intervention. Peripheral blood and hypothalamus were collected at the end of the experiment. The serum leptin level was measured by ELISA method, and the expression of OB-R and POMC in hypothalamus was detected by RT-qPCR method. Methylation level of OB-R and POMC promoter detected by methylation speci fic PCR.Results:Our results revealed that Wendan Decoction (温胆汤) could effectively reduce body weight, the body weight in the drug observation group was signi ficantly lower than that in the model control group, and the difference between the two groups was statistically signi ficant. The level of leptin in peripheral blood and the expression of OB-R in hypothalamus were consistent with the trend of body weight. The heavier the weight, the higher the level of leptin and OB-R. Wendan Decoction (温胆汤)could reduce the level of leptin and OB-R in peripheral blood while reducing body weight. Compared with the model control group, the difference was statistically signi ficant. The heavier the weight, the lower the level of POMC expression. Wendan Decoction (温胆汤) could increase the expression of POMC while reducing body weight, which had statistical signi ficance compared with the model control group. The methylation status of OB-R and POMC promoter in hypothalamus was not signi ficantly associated with obesity induced by high fat diet, and Wendan Decoction (温胆汤) had no effect on methylation status.Conclusions:Wendan Decoction (温胆汤) could inhibit obesity by affecting the expression of leptin receptor and POMC mRNA in hypothalamus to a large degree. There was no obvious change about the methylation status of the gene promoters of leptin receptor and POMC in obese rats fed with high fat diet. And it was not related to the change of methylation status of gene promoter about the mechanism of Wendan Decoction (温胆汤).

KEYWORDS Wendan Decoction (温胆汤); Proopiomelanocortin; Obesity; Leptin resistance; Methylation

INTRODUCTION

Wendan Decoction(温胆汤), as a classic prescription of traditional Chinese medicines to dissipate phlegm, can play a certain role in reducing weight in patients with obesity phlegm dampness syndrome[1-3]. But its mechanism is not yet clear.During the formation of obesity, the leptin produced by the body can reduce the energy intake and inhibit the formation of obesity through the OB-R (Obese Gene Receptor, OB-R) in the hypothalamus and its subsequent pathway. However, the level of leptin in the peripheral blood of obese patients tended to increase, showing hyperleptinemia. It can be seen that the binding of leptin and OB-R does not play a role in regulating energy metabolism, and its mechanism is abnormal with OB-R or subsequent pathway molecules causing disability of leptin-OB-R regulatory pathway. It is related to LR (leptin resistance, LR)[4,5]. Previous other studies had shown that epigenetic mechanisms, represented by gene methylation are involved in the occurrence and development of obesity[6-8]. Among them, methylation of leptin receptor and POMC (proopiomelanocortin,POMC) promoter in hypothalamus participated in the regulation of appetite by affecting the expression of their own genes, and then affected the formation of obesity[9,10]. We believe that the syndrome formation of many chronic diseases may be related to epigenetic mechanism. And whether the overall toning effect of traditional Chinese medicine can play a role by epigenetic mechanism is worthy of research direction[11,12]. Therefore, this research firstly analyzed the leptin levels on peripheral blood,the expression of leptin receptor and POMC genes to explore the leptin resistance mechanism of Wendan decoction of the high-fat feeding obesity in rats.It also analyzed the promoter methylation status of leptin receptor and POMC genes in diet-induced obesity rat, and discussed the in fluence of Wendan Decoction (温胆汤) that leptin resistance was related to change of the related gene promoter methylation status.

MATERIALS AND METHODS

Animals and Feed

60 male SPF SD rats [(60±10) g] were purchased from Hunan SJA Laboratory Animal Co.,Ltd [Certificate of quality: SCXK (XIANG) 2013-0004]. High fat feed (100g basic feed with lard 15g, egg yolk 10g, whole milk powder 15g, pig bile salt 0.5 and concentrated cod-liver oil) was made into granular medicine with B class by Hunan SJA Laboratory Animal Co., Ltd [Certificate of quality:SCXK (XIANG) 2014-0002].

Medicines

Wendan Decoction (温胆汤) consists of the following drugs.Citri Reticulatae Pericarpium(陈皮) 10g,Pinelliae Rhizoma(半夏) 10g,Poria(茯苓) 10g,Glycyrrhizae Radix Et Rhizoma(甘草) 3g,Bambusae Caulis In Taenias(竹茹) 10g,Aurantii Fructus Immaturus(枳实) 10g,Zingiberis Rhizoma Recens(生姜) 5 slices,Jujubae Fructus(大枣), the above herbs were boiled with water according to the method of clinical decoction, 30 mins each time,combined with 2 times of medicine solution, filtered,condensed to 1:1 solution at low temperature in water bath. 1 ml liquid medicine contains 1 gram of crude medicine. After aseptically sealed, it was stored in a refrigerator at 4 degrees Celsius.

Reagents and Instruments

Leptin ELISA kit, Andygene, 96T (No.AD20180109). YBR Green PCR kit, KAPA Biosystems (KM4101). Reverse transcription kit,TAKARA (639505). DNaseⅠ, Fermentas (AM2295).Trizol, ambion company (15596026). Blood/tissue/Cell Genome extraction Kit, TIANGEN (DP304-03).DNA heavy sulfite transformation kit, TIANGEN(DP215).

Enzymatic labeling instrument, US BioTek,instrument Model (Synergy2).Plate washing machine, Finland Thermo Labsystems, instrument Model (AC8). Micro high speed centrifuge, domestic,instrument type (TG16W). Water insulated incubator,domestic, instrument type (GNP-9080). Pure water instrument, Sichuan superior company, instrument model (ZYTEST-I-20L). Fluorescence quantitative PCR instrument, BIO-RAD, Real-Time System. Gel imaging system, BIO-RAD, Universal Hood II.

Modeling and Intervention Methods

SD rats were divided into model group and normal control group after 7 days of adaptation in a shielded feeding environment. The model group was fed with high fat diet and the normal control group was fed with normal diet for 6 weeks. The model group was fed with high fat diet and the normal control group was fed with normal diet for 6 weeks.The rats in both groups were weighed every week,and then according to the success criteria of obesity model after the establishment of the model (SD rats weighing more than 20% of the rats fed with normal diet were obese rats refer to Chandler PC, 2005).Finally, 16 rats in the model group and 8 rats in the normal control group were included. Animal care and experimental procedures used in the current study were approved beforehand by the Institutional Animal Care and Use Committee of Jiangxi University of Traditional Chinese Medicine (Approval No. JZLISC2018-028).

Rats in the model group were randomly divided into blank control group and drug observation group.Rats in the normal control group were included,with 8 rats in each group.The observation group was given Wendan Decoction (15 g/kg of Wendan Decoction was administered lavage, and the dosage of Wendan Decoction was determined according to the body weight of rats[13]). The blank control group and the normal control group were given clean drinking water once a day for 6 weeks.The whole process of animal experiment followed the national guidelines for the management and use of experimental animals and the relevant ethical regulations of experimental animals.

Detection Indicators and Methods

Body mass

The rats in each group were weighed once a week.

Leptin level in peripheral blood

After the intervention, rats in each group were anesthetized by intraperitoneal injection of 2%sodium pentobarbital (45mg/kg). After anesthesia,abdominal aorta was dissected and exposed. Blood was intubated with 5 ml of blood vessels. Serum was separated after blood coagulation and leptin level was detected by ELISA.

The main steps were carried out according to the instructions, including the addition of dilution holes, blank holes and sample holes to be measured; After sealing the plate with film, it was incubated at 37 ℃ for 30 minutes, washed with detergent, repeated five times, patted dry; 100 UL HRP enzyme was added into each pore. In addition to the blank pore, the pore of enzyme label plate was sealed with sealing plate membrane, incubated and was hed; The colour reagent was first added into each hole, and the colour was developed at 37 ℃ for 15 minutes; After 15 minutes, 50 ml termination solution was added to each pore to terminate the reaction; Enzyme labeling plate was put into the detection tank of the enzyme labeling instrument, and the wavelength of 450nm was set. OD value of each pore was determined according to the procedure of the enzyme labeling instrument; Linear regression equation formula of standard curve was calculated by taking standard concentration as ordinate and OD average value as abscissa. The OD value of sample was substituted for its formula, and the concentration of sample was calculated, then multiplied by dilution multiple, that is, the actual concentration of sample.

Expression of OB-R and POMC RNA.

After anesthesia, rats were exposed to the hypothalamus, and the hypothalamus tissues were carefully separated by dissecting scissors. Some of the tissues were ground up and RNA was extracted.The expression of OB-R and POMC molecules was detected by real-time fluorescence quantitative RTPCR. The experimental steps were carried out by conventional methods. Including: Primer3 software was used to design primer sequence, which was synthesized by Nanjing Kingsley Biotechnology Co.,Ltd. The primer sequence is shown in Table 1 below.

Table 1. RT-PCR Primers of OB-R and POMC Genes

After the sample was fully cracked, RNA solution was extracted and collected, quantified by spectrophotometer, and the total amount of RNA extracted was estimated to be 10ug. RNA was stored at ultra-low temperature to prevent degradation. The reaction system was constructed to synthesize the first chain of the DNA, and the prepared DNA was amplified by PCR. Reaction procedure: 95 ℃, 3 min; 95 ℃, 5 sec; 56 ℃, 10 sec;72 ℃, 25 sec; 39 cycles; 65 ℃, 5 Sec; 95 ℃, 50 Sec. Real-time PCR experiments were carried out according to the instructions of the instrument. Data were collected and analyzed by the instrument's own software. The expression level of RNA was measured using the 2-△△Ctrelative quantification method.

Methylation of promoters of OB-R and POMC genes in hypothalamus

DNA was extracted from some hypothalamic tissues and methylation-specific PCR was used to detect the methylation of promoters of OB-R and POMC genes.

The PCR primers were synthesized by Nanjing Kingsley Biotechnology Co., Ltd. as following Table 2.

Table 2. Methylation-Speci fic PCR Primers of Promoters of OB-R and POMC Genes

DNA extraction kit was used to extract tissue genome, bisulfite reaction system was prepared in 200 ul centrifuge tube, bisulfite transformation was carried out on PCR instrument, and then PCR amplification was carried out. Reaction procedure:95 ℃,5min; 95 ℃,10sec; 56 ℃,10sec; 72 ℃,10sec;30 cycles；72 ℃,5min; Stored at 4 ℃. After the PCR experiment, gel electrophoresis experiments were carried out, and 120V voltage, 25min electrophoresis and electrophoresis were used to complete the imaging.

Statistical Methods

In the IBM SPSS SStatistics19.0.0 containment., metrological data are expressed by mean number±standard deviation (x-±s), and independent samplettest was used to compare the mean of two groups. Single factor analysis of variance was used to compare the mean of multiple groups. LSD test was used for homogeneity of variance, and Tamhane test was used for statistical test for variance difference. Fisher exact test method was used for counting data.P＜0.05 is showed signi ficant difference.

RESULTS

Animal Weight

As shown in table 3, the body weight of the model group (including the blank control group and the drug observation group) was significantly different from that of the normal group after 6 weeks of high-fat diet feeding. According to the obesity rate = (model group-normal group) / normal group* 100%, the obesity rate of this model =(423.87-343.35) / 343.35*100% = 23.45%,suggesting that the obesity model was successful.After 6 weeks of drug intervention, there was signi ficant difference between blank control group,normal control group and drug observation group(P=0.001, 0.003), while there was no significant difference between drug observation group and normal control group (P=0.531), suggesting that Wendan Decoction could effectively reduce the weight of obese rats.

Table 3. Modeling and Body Weight After Intervention of Rats in Each Group

Leptin Level in Peripheral Blood

As shown in table 4, the leptin level in blank control group was the highest, followed by drug observation group and normal control group. The blank control group was significantly different from the drug observation group and the normal control group (P=0.000,0.000); Leptin in the drug observation group was higher than that in the normal control group, and there was significant difference between the two groups (P=0.000). These results suggested that leptin levels in peripheral blood of rats increased after weight gain, but decreased after intervention with Wendan Decoction.

Table 4. Leptin Levels in Peripheral Blood of Each Group

Hypothalamic OB-R, POMC Expression

As shown in Table 5, the expression of OB-R was highest in blank control group, followed by drug observation group and normal control group. There were statistical differences between blank control group, drug observation group and normal control group (P=0.001, 0.000). These results suggested that the OB-R level of rats increased after weight gain, and Wendan Decoction could reduce the expression of OB-R after intervention.

Table 5. Expressions of OB-R and POMC in Each Group

POMC expression was the highest in the normal control group, followed by the drug observation group and the blank control group.There were significant differences between the blank control group, the drug observation group and the normal control group (P=0.001, 0.005);There were also statistical differences between the drug observation group and the blank control group(P=0.000). These results suggested that POMC expression decreases after weight gain in rats,and Wendan Decoction could increase POMC expression after intervention.

In addition, as shown in Figure 1 and 2,the expression of leptin in peripheral blood,hypothalamus OB-R and POMC were positively correlated with body weight, while the expression of POMC in hypothalamus was negatively correlated with body weight.

Figure 1. The Relationship Between Leptin Levels in Peripheral Blood and Body Weight in Each Group

Figure 2. The Relationship Between the Expression of OB-R and POMC in Hypothalamus and Body Weight in Each Group

Methylation of Promoters of OB-R and POMC Genes in Hypothalamus

As shown in Figures 3 and 4, "U" means nonmethylation and "M" means methylation.

A sample with "U" positive and "M" negative indicated that the sample gene was not methylated;a sample with "U" negative and "M" positive indicated that the sample gene was methylated;a sample with "U" positive and "M" positive also indicates that the sample gene was not completely methylated.

Figure 3, 4 and table 6 showed that the promoter of OB-R gene was methylated in all three groups. The promoter of POMC gene was methylated and incompletely methylated in the normal control group and blank control group, while there was only incomplete methylation in the drug observation group,but there was no signi ficant difference in methylation among the three groups (P=0.494).

Figure 3. Methylation of the Promoter of Hypothalamic OB-R Gene in Each Group

Figure 4. Methylation of POMC Promoter in Each Group

DISCUSSION

OB-R exists in many tissues of animals and can be expressed in brain, heart, placenta, liver,kidney, prostate and testis. OB-R combines with leptin to regulate energy balance and fat storage.Leptin binds to OB-R in hypothalamus mainly through OB-R in glucagon-like peptide-1 neurons to reduce food intake and body mass. The POMC gene are thought to have an effect on eating behavior,while POMC neurons and POMC gene products in hypothalamus are part of the signaling pathway of leptin action[14,15]. The results showed that the leptin level in peripheral blood of rats fed with high fat diet was higher than that of normal rats, and the OB-R level in hypothalamus was also higher than that of normal rats. This indicated that obese individuals would compensatively increase leptin secretion with the increase of body weight. The combination of leptin in peripheral blood and OB-R in hypothalamus would stimulate the central system, reduce appetite feedback and inhibit obesity production. Further studies have shown that the decrease of POMC mRNA expression in hypothalamus of obese rats may be one of the mechanisms of leptin and OB-R up-regulation while obesity continues to develop,namely leptin resistance, which is the same as previous studies[16,17].

Table 6. Methylation Status of Promoters of OB-R and POMC Genes in Each Group

Wendan Decoction (温胆汤) is a famous prescription for eliminating phlegm in traditional Chinese medicine. It is composed of Fuling (Poria),Banxia (Pinelliae Rhizoma), Zhishi (Aurantii Fructus Immaturus), Chenpi (Citri Reticulatae Pericarpium),Zhuru (Bambusae Caulis In Taenias) and Gancao(Glycyrrhizae Radix Et Rhizoma), etc. It has the functions of clearing away heat and resolving phlegm, regulating qi and strengthening spleen. It can be used for treating obese patients with phlegm.This study suggested that Wendan Decoction could inhibit the development of obesity in rats fed with high-fat diet, and its mechanism might be related to the increase of POMC gene expression in hypothalamus. The study also showed that with the weight loss of obese rats, the levels of leptin in peripheral blood and OB-R in hypothalamus showed a downward trend, indicating that Wendan Decoction(温胆汤) inhibited the leptin resistance mechanism of obese rats through POMC pathway, and made the negative feedback regulation mechanism of leptin and OB-R played a normal role.

Modern genetic studies have shown that complex diseases such as obesity are the result of the interaction of genetic and environmental factors.Genetic factors can not explain all the causes of obesity. Epigenetics, which is closely related to environmental factors, plays an increasingly important role in obesity. The epigenetic mechanism does not change the gene sequence, but modifies the gene by methylation and acetylation under the internal and external environment[18]. This modification can be congenital or induced,which plays an important role in regulating gene expression. Studies have found that some obesityrelated genes undergo epigenetic changes such as methylation, leading to abnormal gene expression, metabolic disorders, and ultimately obesity[19]. However, whether changes such as gene methylation are involved in the occurrence and development of obesity and whether they can become a link of drug action need to be further studied[6]. Some studies have shown that the expression of OB-R in hypothalamus of food-borne obesity model mice is higher than that of normal mice, but the methylation level of CpG locus in promoter region of OB-R gene has no significant difference between food-borne obesity group mice and normal mice[20], which is the same as the results of this study. Overnutritional diet can cause obesity in neonatal rats. The promoter region of POMC gene in hypothalamus is highly methylated and the expression of POMC gene is down-regulated. The level of methylation is negatively correlated with the expression of POMC gene[21]. Hypermethylation of POMC exon 3 interferes with the binding of transcriptional enhancer P300, reduces the transcriptional expression of POMC, and is associated with the risk of obesity[22]. The results also showed that POMC expression in hypothalamus of rats fed with high-fat diet decreased, and its promoter region showed methylation status. There was no statistical difference between normal diet rats and high fat diet rats. It was dif ficult to explain whether the down-regulation of POMC expression was related to the increase of methylation in the promoter region of the gene, but whether there was an up-regulation of methylation in the exon of POMC was not involved in this study.

CONCLUSIONS

The results of this study suggest that Wendan Decoction (温胆汤) may inhibit obesity leptin resistance through POMC. Although the existing research showed the methylation changes of OB-R and POMC genes in obesity, and the methylation genes could be intervened as drug targets. But it also had the results which do not support this conclusion. The results also showed that the promoter methylation status changes of OB-R and POMC genes and obesity caused by high-fat diet had no clear correlation, nor the intervention effect of Wendan Decoction (温胆汤). Related gene methylation is involved in the formation of fat, or drug targets, and more research is needed to discuss.