Lycium ruthenicum Murr. alleviates nonalcoholic fatty liver in mice

Abstract Oxidative stress and inflammation contribute to hypertriglyceridemia‐induced nonalcoholic fatty liver disease (NAFLD). Cholesterol‐enriched diets increase the risk of NAFLD. Lycium ruthenium Murr. (LRM) contains water‐soluble antioxidant proanthocyanidins. Whether Lycium ruthenium Murr. improves NAFLD remains elusive. In this study, we established a model of NAFLD‐induced by cholesterol‐enriched high‐fat diet (western diet) in ApoE −/− mice; oxidative stress and inflammation were examined and intervened by supplement of Lycium ruthenium Murr. (LRM) extracts. LRM supplement did not influence body weight gain, food intake, and lipotoxicity of mice. LRM supplement significantly alleviated triglyceride accumulation in liver, with reduced inflammation, elevated GSH‐Px activity, and reduced MDA levels. The expression of fatty acids oxidative gene Scd1 was significantly increased, and fatty acids synthesis‐related gene Pparγ was dramatically downregulated on mRNA level in liver of mice with LRM supplement. These data demonstrated that LRM supplement decreased ROS production and inflammation, increased fatty acids oxidation, and reduced fatty acids synthesis in liver, leading to ameliorate the development of NAFLD induced by high western diet. Thus, oxidative stress and inflammation also are involved in the pathogenesis of western diet‐induced NAFLD, which is independent of obesity.

simple steatosis and increases liver injury; the second hit is primary lipotoxicity caused by inflammation and oxidative stress within hepatocytes (Tu et al., 2017;Xu et al., 2015). However, although triglycerides accumulate in hepatocytes, they do not accumulate in the arterial wall.
Recently studies have showed that hypercholesterolemia increased the risk of NAFLD (Ma et al., 2008;Tous, Ferre, Camps, Riu, & Joven, 2005). In addition, because cholesterol can be accumulated in arteries and liver, NAFLD induced by cholesterol overload accompanies by atherosclerosis in arteries. However, it is unknown if cholesterol-induced hepatic steatosis for further injury is similar to triglyceride-induced fatty liver. Oxidative stress and inflammation are recognized as major causes of the pathogenesis of NAFLD in obese patients (Roskams et al., 2003;Serviddio, Bellanti, & Vendemiale, 2013). Inflammation also aggravated the hypercholesterolemia-induced NAFLD progress (Kim et al., 2014). However, whether oxidative stress also contributes to the pathogenesis of cholesterol-induced NAFLD remains uncertain.
Here, we investigated the protective effect of LRM on the development of cholesterol-enriched high-fat diet-induced NAFLD. Our results showed that LRM significantly reduced fatty acids accumulation due to increased oxidation and reduced synthesis, inflammation, and ROS production in hepatocytes, resulting in alleviating nonalcoholic fatty liver disease in ApoE −/− mice, which is independent of obesity. Our results provided an alternative choice for the treatment of NAFLD and a research model for the pathogenesis of NAFLD.

| Crude flavonoids extract and antioxidant activity assay
The fruit of Lycium ruthenium Murr. (LRM) was purchased from Qinghai. The fruit of Lycium ruthenium Murr. was oven-dried at 50°C and subsequently crushed into powder. Then, the powder was suspended in 75% ethanol (60°C, 1:20 w/v) for 30 min to remove the fruit residues, protein, and polysaccharide sediment through filter paper (Lumeng, Bodzin, & Saltiel, 2007). All extraction solutions were concentrated with a rotary evaporator under 60°C, then were dissolved in distilled water. Total flavonoids content was determined by spectrophotometer (El-Haci et al., 2013). Each sample (1 ml) was mixed with 0.5 ml of NaNO 2 solution (5%). After 6 min, 0.5 ml of Al (NO 3 ) 3 solution (10%) was added into the mixture and allowed to stand for another 6 min. Then, 2 ml of NaOH solution (4%) was added to the mixture and stood for another 15 min. Absorbance of the mixture was determined at 510 nm versus water blank. A calibration curve was performed in parallel under the same operating conditions with rutin as a positive control. The sample was measured 3 times to obtain the average value. Results were presented as rutin equivalent per gram of dry extract (mg RU/100 g dry).
The effect of LRM on scavenging DPPH radical was determined by the modified method described as previous report (Li et al., 2009). Briefly, 2 ml of DPPH solution (2 mM dehydrated alcohol) was added to 1.0 ml of crude flavonoids in water. The mixture was shaken and stood for 30 min at room temperature in the dark.
The absorbance was measured at 517 nm with a UV-vis spectrophotometer. The DPPH radical scavenging effect was calculated as follows: DPPH scavenging effect (%) = (A 0 − (A − Ab))/A 0 × 100%, where A 0 is the A517 of DPPH without sample, A is the A517 of sample and DPPH, and Ab is the A517 of sample without DPPH.
Hydroxyl radical scavenging activity of the crude flavonoids from LRM was determined as previous report ( Ax is the A510 of sample, and Ax 0 is the A510 of sample without H 2 O 2 .

| Animals and assays
All

| Statistical analysis
All data were presented as mean ± SEM, and differences were assessed by Tukey's multiple comparisons after analysis of variance (ANOVA). Statistical significance was defined as *p < .05. **p < .01.

| Lycium ruthenium Murr. extracts have strong scavenging free radical activity
Since flavonoids are natural antioxidant with strong free radical scavenging activity, it promoted us to examine the antioxidant activities of LRM extracts by the DPPH assay which is widely used to evaluate the free radical scavenging activity of plant extraction.
As shown in Figure 1a, the scavenging activity of DPPH radical was significantly enhanced with increasing concentration of extracts and reached a peak when the concentration was more than 0.06 mg/ml.
Additionally, the antioxidant activity of substance is also determined by scavenging the hydroxyl free radical. As shown in Figure 1b, LRM extracts exhibited high hydroxyl radical scavenging activities in a dose dependent manner. Taken together, these observations showed that LRM extracts have strong free radical scavenging and antioxidative activity.

| Mice body weight gain remains unchanged with LRM supplement
To investigate the effect of LRM on the pathogenesis of NAFLD,

| LRM supplement ameliorates the development of NAFLD
The liver morphology, weight, and indices of liver/body weight were similar in mice on WD with LRM supplement and mice on WD (Figure 3a

| LRM reduces inflammation and ROS production
Chronic inflammation is associated with the progression of NAFLD toward higher risk cirrhotic states (Wijesundera et al., 2016). To investigate whether LRM alleviated the pathogenesis of NAFLD by reducing inflammation, we first examined the expression of pro-inflammatory genes. The mRNA level of Tnf-α was remarkably decreased in mice with LRM supplement, indicating an antiinflammatory effect of LRM (Figure 5a). However, the expression of pro-inflammatory gene Il-6 in liver was not significantly changed by LRM supplement (Figure 5b). The transcript level of anti-inflammatory Il-4 gene was dramatically increased in mice on WD with LRM supplement compared with WD-fed mice (Figure 5c), while Il-10 mRNA level was similar in mice with or without LRM supplement ( Figure 5d). These results indicated that LRM supplement improved inflammation by regulating Tnf-α and Il-4 expression.
Reactive oxidative species (ROS) are strongly associated with the progress of hepatic steatosis (Videla et al., 2004). To address this, we first measured the GSH-Px activities and MDA levels, which were responsible for oxidative stress in tissue. We found that mice with LRM supplement showed dramatically elevated GSH-Px activities compared with mice on WD (Figure 5e). MDA levels in liver were significantly higher in mice on WD than NC. LRM supplement dramatically reduced MDA levels in mice (Figure 5f), indicating an inhibitory effect of LRM on ROS production. Together, these observations suggested that LRM supplement reduced oxidative stress induced by cholesterol-enriched high-fat diet.

| D ISCUSS I ON
In the study, we found that oxidative stress and inflammation are associated with cholesterol-induced NAFLD in ApoE −/− mice.
Antioxidant LRM supplement remarkably slowed down the pro- Consistently with previous studies that fat accumulation in the liver was independent of body mass index and intra-abdominal and overall obesity (Seppala-Lindroos et al., 2002).
All these results revealed that LRM ameliorates the pathogenesis of hepatic steatosis induced by cholesterol-enriched high-fat diet, coupled with reduced ROS production and inflammatory levels.
In conclusion, our results indicated that oxidative and inflammation are involved in the pathogenesis of cholesterol-enriched high-fat diet-induced NAFLD, which is independent of obesity.

Reduction of oxidative stress and inflammation by antioxidant
LRM suppressed the progression of NAFLD-induced by cholesterol-enriched high-fat diet. However, the really active components in the crude flavonoids still need to be identified by further experiments. Further experiments are also required for the elucidation of the underlying mechanism of LRM ameliorating the pathogenesis of hepatic steatosis.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human testing, and the animal study's protocols and procedures were ethically reviewed and approved by the Jiangnan University.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.