The treatment of obesity is confronted with a stern challenge at present. With the commercial withdrawal of Sibutramine and Orlistat, no effective and low-toxicity new weight-reduction drug has been developed and marketed. Our and other studies have demonstrated that Matrine could reduce the body weight and Lee’s indexes of obese rats, as well as fasting insulin, TG and cholesterol levels . By taking advantage of the TCM theories, our study not only holistically confirmed that Matrine could reduce the body weight and abdominal fat weight of obese rats, decrease the size of fat cells and increase the HDL-C level but gained insights into the mechanism of Matrine in affecting the adipose tissue of obese rats. Based on our previous work, the present study, for the first time, used the thin-film dispersion method to successfully prepare a nano NET emulsion. The obtained TF carry negative charges and are small in size and well distributed, with good entrapment efficiency and stability. The result of external application showed that it was able to relieve the pathological change of fatty liver, reduce the peripheral fat content, increase serum HDL-C, and reduce TG level. In addition, it could also down-regulate the G0S2 protein expression in the adipose tissue of obese rats and up-regulate the ATGL protein expression. These mutually antagonistic effects work together to reduce the body weight of obese rats. There is no similar report available in the literature. The mechanism of NET in the prevention and treatment of obesity needs to be further studied, knowing that regulation of G0S2 and ATGL may prove to be a new strategy for the treatment of obesity.
The development of obesity is attributed to multiple factors responsible for disordered homeostasis of fat metabolism, increased fat synthesis and decreased fat decomposition in the body. Studies have demonstrated that multiple factors participate in and regulate fat metabolism. ATGL and G0S2 are believed to play an important role in regulating fat metabolism. ATGL is a key enzyme in fat metabolism, and is expressed mainly in murine white and brown fat tissues, and secondarily in the cardiac muscle, skeletal muscle and testis [5, 9]. ATGL mainly hydrolyzes triglyceride into diglyceride, and therefore ATGL and HSL are believed to be a common rate-limiting enzyme for the reaction of fat decomposition. The adipose tissue was increased remarkably in ATGL knockout mice, presenting as TG accumulation in multiple tissues, indicating that ATGL is a rate-limiting enzyme of TG hydrolysis, playing an important role in fat mobilization in fat tissues [26–28]. Other studies also reported that G0S2 is a negative regulator of ATGL . Zandbergen et al. reported that the level of G0S2 mRNA expression was the highest in fat tissues, and was up-regulated in the process of preadipocyte differentiation into fat cells . In vitro and cell-based studies on G0S2 function showed that G0S2 directly binds to ATGL, thus decreasing the ability of ATGL-mediated fat decomposition by inhibiting the activity of TAG hydrolase .
In the present study, we established a rat model of alimentary obesity induced by feeding the animals with well-prepared high-fat chows for two weeks. After 6-week feeding, the body weight of the animals in the model group was 20% heavier than that in the normal group. In addition, both wet weight ratio and body lipid ratio of the internal organs of the animals in the model group were increased. HE staining also showed that fat cells became significantly larger as shown by optical microscopy. All these indicate the successful establishment of the obesity model. Animal studies have demonstrated that the expression of ATGL mRNA was down-regulated markedly in db/db and ob/ob obesity animal models, suggesting that the development of obesity may be associated with the decreased level of ATGL [6, 25]. The result of the present study showed that the expression level of ATGL protein in the model group was slightly lower than that in the normal group, which may be the reason for the increased fat synthesis and excessive accumulation of fat in the body, resulting in obesity. Our finding is grossly consistent with the results of other studies.
The present study holistically confirmed that NET could reduce body weight and abdominal fat weight of obese rats. The biochemical parameters also showed that TG was decreased and HDL-C was increased after topical treatment with NET. Compared with the model group, the equivalent diameter, circumference and area of fat cells in the intervention group were also decreased significantly. Pathological observation showed that the liver tissue was normal, suggesting that NET emulsion may affect the process of energy consumption in fat metabolism. In summary, ATGL and G0S2 play an important role in energy metabolism of the body. In the present study, we used the WB technique to further observe its impact on ATGL and G0S2 protein expression in the adipose tissue of obese rats. The result showed that NET was able to down-regulate G0S2 protein expression in the adipose tissue of obese rats, and up-regulate ATGL protein expression to exert its weight-reduction effect. The above pharmodynamic study demonstrated that GOS2 and ATGL could regulate fat metabolism, which is consistent the previous studies [31–33]. We also testified the effect of G0S2 in attenuating the action of ATGL in rats in vivo. The blood biochemical parameters, pathological observation of the fat and liver tissue, and molecular biological parameters obtained in our study indicate that NET emulsion had a good weight-reduction therapeutic effect equivalent to that of oral emodin, which is also consistent with our previous study . But the therapeutic effect of the external NET emulsion is better than that of massage in reducing body weight.
Emodin is a kind of water soluble molecules and hard to be entrapped. Transdermal drug delivery system has been accepted as potential non-invasive route of drug administration, with advantages of avoidance of the first-pass metabolism, sustained therapeutic action and better patient compliance, though, its prevalent use is restricted due to excellent impervious nature of skin. It is the greatest challenge for researchers to surmount the inherent limitations imposed by stratum corneum of skin, for enhanced transdermal drug delivery to achieve systemic therapeutic concentration . Transfersomes have shown immense potential in drug delivery across the skin . Since the transfersomes has high hydrophilicity and flexibility. In the role of hydration transfersomes could across l/10 ~ l/4 of itself diameter of the channel, made loaded drugs through the skin barrier and even enter into the blood circulation. Thus, preparing NET is a good way to improve its entrapment efficiency.
The preparation method by reverse-phase evaporation was simple and had high encapsulation efficiency, easy for scale up preparation and research . We choosed the deoxycholic acid salt as surfactant, when the weight ratio of deoxycholic acid salt and phospholipids was 1:8, the NET had well stability and high entrapment efficiency. The experiment indicated that encapsulation efficiency was the highest when the weight ratio of cholesterol and phospholipids was 1:2, the amount of emodin was 10 mg, the pH value of PBS was 7.2 and the ether volume was 25 mL. RP-HPLC is the most widely used separation technique for this application owing to its simplicity and general applicability to emodin type alkaloids , which could determine entrapment efficiency accurately as well as emodin quantity. The average entrapment efficiency of NET was 61.46 ± 0.25% by RP-HPLC determination. Zeta potential is one of the important indexes of the surface charge of ion, and also affected the stability of transfersomes suspension liquid [39, 40] transfersomes with negative potential can reduce mutual gather and fusion, increase the stability. The Zeta potential of NET prepared in this experiment was −7.94 mV. And the particle size of NET with negative charge was small and uniform, and had some stability. The above methods for preparation of NET and determination of its physicochemical properties, provide excellent separation and good precision, and are relatively straight forward and reliable for both chromatographic conditions and sample preparation. Furthermore, the study of NET was not comprehensive, and need further investigation.
The results of the present study may also provide some inspirations and references for further research on the preparation of nano materials and treatment of obesity, and for the development of new weight-reduction drugs as well.