The present study examined the anti-obesity potential of PNS and PNE, and explored the mechanism of obesity-related ER stress and cell apoptosis in DIO mice. Positive results were obtained: (1) chronic treatment of PNE significantly improved the obesity-induced pathological changes such as avoiding an excessive fat accumulation, decreasing blood lipid levels and resisting fatty liver but PNS at the same dosage showed moderate anti-obesity effects; (2) PNS and PNE inhibited obesity-related ER stress and the associated apoptosis and inflammation in eWAT.
In the past decades, the global epidemic of obesity has attracted more and more attention. Metabolic disturbance is aggravated by overnutrition and/or modern sedentary lifestyle and other risk factors consist of environment and genetics [24]. It is well known that obesity causes many health problems, including high blood pressure, fatty liver, diabetes, and increasing the risk of cancers [25]. The most intuitive manifestations of obesity are weight gain and massive accumulation of fat. Expansion of eWAT quickly responds to obesity in body with increasing in adipocyte number and cell size [26]. In previous study, PNS was found to reduce body weight and fat mass in DIO mice [27]. However, our current data showed that PNS could not avoid these adverse consequences of HFD. These contradictions might be due to the different treatment conditions. In our present study, we treated the mice with a low dosage at 20 mg/kg/day and the HFD was the type with 45% fat; while PNS administered in that previous study was high dosage, 400 and 800 mg/kg/day, and HFD with 60% was used. Importantly, we provided the novel findings that the whole extract PNE was more potent than PNS at the same dosage to reduce the visceral white adipose tissues, eWAT and rWAT, accompanied by reversal of adipocyte hypertrophy.
Obesity detrimentally affects lipid metabolism including triglyceride and cholesterol productions. When the storage capacity of adipose tissue reaches saturation, ectopic fat deposition and increased circulating free fatty acids (FFA) will occur due to the extravasation of excess fatty acids from adipocytes, resulting in lipotoxicity to various organs or tissues [28]. Non-alcoholic fatty liver disease can be found during the progression of obesity and typical features are increases in lipid content and liver damage. Herein, both PNS and PNE were able to ameliorate the lipid metabolism and fatty liver: (1) lowering the levels of triglyceride and cholesterol in plasma and liver tissues to different extents; (2) greatly inhibiting hepatic fat accumulation; and (3) diminishing liver function-related indicators ALT and AST. These results are consistent with the recent study on PNS [29]. Amelioration of fatty liver is beneficial to reduce the increase of FFA-induced acyl coenzymes A (CoA) [30], and ultimately prevent the synthesis of endogenous cholesterol [31, 32].
Obesity will lead to cell hypoxia because of limited angiogenesis and excessive adipose tissue, and thereby trigger inflammation and apoptosis [10]. Proinflammatory factors are released in hypertrophic adipose tissue, as for example, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) [33]. Proinflammatory macrophages are recruited into fat depots by inflammatory factors to form crown-like structures, which are well recognized as the histologic hallmarks of inflammatory and dead adipocytes [34]. The crown-like structures were present in eWAT from DIO mice but were absent in samples from PNS- and PNE-treated groups. This result implies that PNS and PNE reduced cellular inflammation and apoptosis in adipocyte hypertrophy, which were further verified by examining protein expressions of related signaling pathways.
In obesity, adipocyte hypertrophy and massive lipid accumulation are associated with ER stress. ER stress increases macrophage infiltration, triggering inflammation and apoptosis in adipocytes [34, 35]. Upon ER stress, GRP78/BiP, an ER chaperone dissociates from the three ER stress sensors, PERK, IRE1 and ATF6 to activate the downstream signaling cascade. Both JNK and p38 MAPK are known to be downstream targets of IRE1 pathway; and activation of p38/JNK signaling pathway mediates not only apoptosis but also inflammation. All the three pathways ultimately induce the activation of CHOP and caspase-3 to initiate apoptosis [36, 37]. As reported previously, HFD significantly increased the expressions of ER stress-responsive proteins such as CHOP and GRP78 in eWAT [13, 38]. There is also convincing evidence that adipose tissue from DIO mice had enhanced phosphorylation of JNK [39]; and inhibition of JNK activity could reduce adipocyte apoptosis [40]. Moreover, CHOP is linked to inflammation. HFD-induced macrophage infiltration was improved in CHOP−/− mice [41]. Similarly, we found that expressions of GRP78, cleaved (active) ATF6, phosphorylated JNK at Thr183/Tyr185, phosphorylated p38 at Thr180/Tyr182, CHOP and caspase-3 were upregulated in the eWAT from obese mice, revealing the occurrence of ER stress and the associated inflammation and apoptosis. These proteins in eWAT were effectively downregulated by PNS and PNE treatments. In other cell type like cardiomyocytes, PNS [42] and notoginsenoside R1 [43] protect against ER stress-related signaling pathways. The anti-inflammatory potential of PNS has been widely demonstrated [44, 45], and notoginsenoside R1 can suppress p38/JNK pathway to protect PC12 cells from neurotoxicity [46]. In line with the previous studies, we provided the novel findings that not only PNS but also PNE can inhibit ER stress-mediated inflammation and apoptosis in adipocytes, exerting the anti-obesity effect.