Body and liver weight
Fig 1 (A and B) shows that, as observed in
Fan et al., (2005), lipidation of the liver began after 8 weeks on a high-fat diet, progressing to steatohepatitis with pericellular fibrosis by week 12. Rats on the high-fat diet also showed a significantly greater increase in body weight compared to those on a general diet after 10 weeks, along with a higher liver weight due to lipidation. The NAFLD + MA and NAFLD + WA groups started showing a significant decrease in body weight after 12 weeks compared with that of the NAFLD groups and the difference increased further until the completion of the experiment. Moreover, the liver weight decreased significantly with changes in body weight. This indicates that, as in
Meng et al., (2019) study, acupuncture can significantly affect the increase in liver weight because of lipid accumulation in the liver caused by a high-fat diet.
TC and TG
NAFLD due to a high-fat diet is characterized by TG accumulation in the cytoplasm of hepatocytes and high levels of TG accumulation lead to a fatty liver, which causes an increase in inflammatory cytokines or tissue necrosis and fibrosis. The damaged liver leads to a release of TC and TG into the bloodstream, increasing these levels. These serum biochemical markers are used in specific and sensitive evaluations of liver damage in preclinical studies
(Palekar et al., 2006; Wilkins et al., 2013).
As depicted in Fig 1 (C and D), a high-fat diet increased the levels of TC in the serum and TG in the hepatic tissue. The NAFLD + MA group had significantly lower TC levels in the serum and lower TG levels in the hepatic tissue than the NAFLD group. Similarly, the body and liver weights in the NAFLD+MA group reflect the relieving and regulating effects of acupuncture in the stages of lipid accumulation. These findings are consistent with those reported by
Wang et al., (2019) and
Zhang et al., (2020), which demonstrated that MA also reduced the elevated TC levels in the liver caused by a high-fat diet.
Histology
NAFLD is defined as the state in which over 5%-10% of the liver cells indicate visible steatosis with no other cause of liver disease
(Bondini et al., 2007). In the hematoxylin and eosin stain, the NAFLD group showed many accumulated lipid droplets, infiltration and liver lobule structure disorder in multiple locations, whereas acupuncture groups showed a remarkable improvement in lipid droplets and infiltration Fig 2(A). Furthermore, in Masson’s trichrome stain, the NAFLD group showed a high rate of lipid droplets compared with that of the control group and fibrosis by infiltration, whereas the acupuncture groups showed a decrease in the area of lipid droplets Fig 2(B). In the Oil red O stain, the control group showed the blue-stained nuclei of liver cells without lipid deposition, whereas the NAFLD group showed diffused lipid droplets stained in red and the nuclei appeared compressed compared with normal cells because of the spread of lipidized cells and the acupuncture groups showed relatively little lipid droplets and more nuclei Fig 2(C). We tracked three indicators, including the symptoms of lipid accumulation in the liver. The histological findings of the liver in this study indicate that, as demonstrated in the research by
Alshehri et al., (2024), the damaged liver exhibits a general expansion of infiltration and fibrosis compared to normal liver. Importantly, the liver in the adequately treated group showed a significant visual improvement in tissue damage alleviation, reinforcing the effectiveness of the treatment. Finally, perilipin is a protein located on the surface of lipid droplets in adipocytes and steroid-producing cells that are involved in lipid storage by reducing lipid degradation in a dephosphorylated state (
Kimmel and Sztalryd, 2016). Our experiment also revealed that perilipin was locally expressed around lipid droplets in the hepatic tissue of the NAFLD model compared with that of the control group, whereas in the acupuncture groups, the expression of perilipin reduced remarkably Fig 2(D).
According to the results of histological analysis in our study, the hepatic tissues and cells in the NAFLD group were lapidated compared with those in the control group and fibrosis due to lipidation of normal cells progressed simultaneously with the occurrence of structural collapse, whereas the acupuncture groups were involved in reducing lipidation-caused fibrosis through structural normalization of tissues by reducing TG level in the tissue and lipidation of liver cells. These results correspond to the above results of the reduced liver weight and lipid-related factors.
Resistin
As a recent therapeutic experimental approach,
Ma et al., (2020) reported that acupuncture improved inflammatory cytokines such as
TNF-α by inhibiting the
NF-κB signaling pathway and lipid metabolism in an NAFLD model. In this study, we further examined how the effect of acupuncture on fat accumulation induced by a high-fat diet shown above could affect
NF-κB p65/TNF-α, which can induce inflammation in the liver. Adipokines secreted by the fat tissues are classified into proinflammatory and anti-inflammatory and the proinflammatory adipokines, such as resistin, promote the development and progression of NAFLD as well as cause inflammation (
Pang and Lee, 2006). Resistin, the inflammatory cell-activating hormone, is known to be mediated by
NF-κB to increase its production through
TNF-α stimulation, inducing inflammation in various cells
(Wen et al., 2021; Chávez-Tapia et al., 2014).
In our study, resistin showed a significant decrease only in the NAFLD + WA group compared with that in the NAFLD group Fig 3(A). These findings suggest that resistin, which activates inflammatory factors that may manifest as fatty deposition in hepatic tissue, can be regulated by WA but is not related to MA.
mRNA expression
TLR4 produces various proinflammatory, antiviral and antibacterial cytokines when it is activated by lipidation of the liver
(Sung et al., 2016). TLR4 is normally expressed in diverse types of cells, such as hepatocytes, monocytes, Kupffer cells and stellate cells
(Broad et al., 2007; Sung et al., 2016). Triggering the
TLR4 pathway leads to the activation of inflammatory cascades, producing various proinflammatory cytokines, including nuclear translocation of
NF-κB and
TNF-α, which causes inflammation and cell death
(Long et al., 2023). Li and Fang (2022) reported the inflammation-regulating action of acupuncture through inhibition of the
TLR4 signaling pathway and the
NF-κB pathway in mastocytes as well.
Our study found that the pro-inflammatory factors
TLR4 significantly decreased in both NAFLD + MA and NAFLD + WA groups Fig 3(B), whereas
NF-κB p65 and
TNF-α significantly decreased only in the NAFLD + MA group Fig 3 (C and D). These results suggest that WA may have a limited effect on the activation of resistin and
TLR4 mRNA, which are inflammation-related factors potentially triggered by fat accumulation in liver tissue resulting from a high-fat diet. Conversely, MA may facilitate the repair of liver tissue damage associated with fat accumulation by activating
TLR4 mRNA, which subsequently regulates
NF-κB p65 and
TNF-α mRNA.