Lingguizhugan decoction (LGZG) is a classic TCM formula, which has been used to treat obesity and hyperlipidemia in recent years, but the related mechanisms by which LGZG regulates lipid metabolism is yet not clear. In this study, we demonstrated that LGZG significantly attenuated HFD-induced fatty liver disease, as measured by body weight, liver index, EFP/BW, liver injury, and hepatic TG. The possible mechanisms might include increasing serum THs and improving beta-oxidation (via modulation of TRβ1 and CPT1A expression), metabolism, and transport (through modulation of SREBP-1c, ACSL, and ApoB100 expression) of fatty acid. Our study, for the first time, revealed the mechanisms through which LGZG regulates lipid metabolism; furthermore, we discovered the herbal combination with the properties of warming yang to relieve water retention in the formula and proposed the biological basis of LGZG conventional effect via further study on disassembled formula. It is of great significance in understanding the classic formulas' conventional efficacy.
NAFL is characterized by the accumulation of TGs, which are formed from free fatty acids (FFA) and glycerol within the hepatocyte. There is a positive TG cycle between liver and adipose tissue in physiological conditions. It can only be exported from the liver in very low-density lipoprotein (VLDL) particles after incorporation into the apolipoprotein (ApoB100) because TG is liposoluble and insoluble in body (an aqueous environment). The ability of synthesizing TG in the liver is greater than that of synthesizing ApoB100 and package VLDL then as a result, NAFL commonly occurs in association with imbalance between production of TG and apolipoprotein synthesis.
FFA and glycerine are hydrolyzed by lipase from TG within the hepatocyte and then released into blood, and FFA generates energy via β-oxidation. FFA is firstly translated to activated acyl-CoA before the catabolic oxidation, and the latter one is freely soluble in water. Thus the metabolic activity of FFA is enhanced by catalyzing acyl-CoA synthetase (ACS).
Our results demonstrated that LGZG, LZG, and LG significantly increased hepatic ACSL (a major isoform of ACS in liver) and ApoB100 in rat models of NAFL. As these three formulas have a common herb Poria and the formula without Poria (GZG) showed no such effect, we suggested that Poria (which is sweet and tasteless in flavor, with a function of drain dampness with bland) is the key factor why a formula relieves water retention. Poria might function through promoting package of VLDL from TG, enhancing aqueous solubility and metabolic activity of fatty acid. The major biological basis of relieving water retention is the high expression of ACSL and ApoB100.
LGZG has the function of warming yang to relieve water retention, and there are similarities between TCM yang-warming and THs function. THs show great importance in metabolic regulation as they significantly promote energy metabolism and fat metabolism, and particularly they improve lipid mobilization, reduce fat storage, and accelerate fatty acid oxidation. Liver is an important target organ where THs execute its function by binding to thyroid hormone receptors (TRs) physiologically, and it also plays an important role in the synthesis, transformation, and inactivation of THs. TRβ
1, a major isoform of TRs in liver, is the major modulator in T3 regulation of cholesterol metabolism which plays a key role in hepatic lipid metabolism [14
]. Preclinical studies have been conducted for TRβ
1 receptor agonists and showed that they reduced plasma cholesterol and TG. In an animal study, a TRβ
1 receptor agonist (M07811) was found to accelerate mitochondria fatty acid oxidation and alleviate hepatic steatosis [16
Studies have demonstrated that TR regulated CPT1 expression by binding to TH-response elements (TRE) in the promoter regions of carnitine palmitoyltransferase-1 (CPT1), a rate-limiting enzyme in hepatic mitochondria fatty acid oxidation [17
]. CPT1 expression is closely related to body fat percentage, and the gene expression is regulated at transcriptional level [21
]. High-CPT1 expression is correlated with high decomposition of fatty acid, low body fat percentage, alleviated hepatic steatosis, and delayed occur of fatty liver [22
]. Three isoforms of CPT1 are currently known: CPT1A, CPT1B, and CPT1C. CPT1A (liver isoform) is mainly expressed in liver, kidney, and pancreas, with the function of fatty acid β
-oxidation regulation [23
]. The CPT1A expression in NAFLD patients is reduced, and hepatic TG is reduced by increasing CPT1A expression [24
]. Hepatic TG level is significantly increased while CPT1A activity is restrained [25
]. Enhanced CPT1A activity is correlated with improved fatty acid β
-oxidation, reduced injury caused by high FFA and TG, and increased TG secretion [26
]. Another study has shown strong interaction between CPT1A and ACSL [27
Hashimoto et al. [28
] demonstrated that T3 significantly reduced mice SREBP-1c expression via TRβ
1, and this suppression might be caused by TRs and LXR (from SREBP-1c) competition for a DNA binding site [29
]. The expression of genes involved in lipid metabolism and glycometabolism is regulated by SREBP-1c, an isoform of sterol regulatory element binding proteins (SREBPs) in liver [30
]. Previous study reported that SREBP-1c regulated synthesis and storage of TG in liver [32
]. SREBP-1c overexpression can cause dyslipidaemia and lead to lipid accumulation and fatty liver. It was confirmed that hepatic fat content in ob/ob mice with a superimposed knockout of SREBP-1 was significantly lower than that in wide-type controls [33
]. SREBP-1c regulates lipid synthesis via transcription regulation of hepatic lipase by changing its mRNA level. Our results demonstrated that LGZG, GZG, and LG significantly increased hepatic TRβ
1 in rat models of NAFLD. As these three formulas have a common herb Ramulus Cinnamomi
(with a function of warming yang for qi activation), while formula without Ramulus Cinnamomi
(LZG) showed no such effect and all four formulas showed beneficial effect on CPT1A, we suggested that Ramulus Cinnamomi
is the key factor why a formula warms yang for qi activation. Ramulus Cinnamomi
might function through increasing THs, hepatic TRβ
1, and CPT1A expression and enhancing fatty acid β
-oxidation. Meanwhile, herbal combination (Rhizoma Atractylodis Macrocephalae
and Radix Glycyrrhizae
) with the property of fortifying the spleen and replenishing qi might strengthen this effect. The major biological basis to warm yang for qi activation is the high expression of TRβ
1 and CPT1A.
In addition, our results demonstrated that LGZG and LG significantly reduced hepatic TG level in rat models of NAFLD, and formulas without Poria (diuresis) or Ramulus Cinnamomi (yang-warming for qi activation) showed no such effect. Based on the theory of syndrome differentiation through formula and correspondence of prescription and syndrome, we proposed that water and dampness retention caused by spleen yang deficiency is the basic TCM pathogenesis of NAFLD, and warming yang to relieve water retention is an effective therapeutic principle in NAFLD preventionand treatment. This reflects the therapeutic idea rooted in Jingui Yaolue: conditions with phlegm and fluid retention should be modulated by drugs with warming nature. The combination of Poria and Ramulus Cinnamomi might be crucial in LGZG, which is established under the therapeutic principle of Warming Yang to Relieve Water Retention. This formula directly supports the idea that there is a harmonious combination of warming tonification and pathological accumulation elimination. The herbal combination of Rhizoma Atractylodis Macrocephalae and Radix Glycyrrhizae enhances the effect of warming yang to relieve water retention.
In summary, our study confirmed that LGZG provided significant beneficial effect on HFD-induced rat models of NAFLD, and the mechanisms underlying the effect of LGZG may include increasing THs and improving fatty acid β-oxidation and metabolism. LGZG might be an alternative therapy for MS, such as NAFLD, based on the data from the present study. However, further clinical trials about LGZG efficacy and studies about other possible mechanisms are warranted.