Alleviative effect of Ruellia tuberosa L. on NAFLD and hepatic lipid accumulation via modulating hepatic de novo lipogenesis in high‐fat diet plus streptozotocin‐induced diabetic rats

Abstract Ruellia tuberosa L. (RTL) exhibits phytochemical activities and has been used as a folk medicine for curing diabetes mellitus in East Asia for decades. This study investigated the effect of RTL aqueous and ethanolic extracts on nonalcoholic fatty liver disease (NAFLD) and hepatic lipid accumulation in high‐fat diet (HFD) and streptozotocin (STZ)‐induced type 2 diabetes mellitus (T2DM) rats. Administration of RTL aqueous extract (RTLW) or ethanolic extract (RTLE) at dosage of 100 or 400 mg/kg body weight for 4 weeks was carried out in HFD/STZ‐induced T2DM rats. Liver weight, adipose (epididymal and perirenal adipose tissues) weight, hepatic triglyceride level, and de novo lipogenesis (DNL)‐associated protein expression were monitored after scarification. The results revealed that RTLW and RTLE reduced relative liver weight and relative fat weights in HFD/STZ‐induced T2DM rats. RTLW and RTLE also ameliorated NAFLD and hepatic triglyceride (TG) accumulation in diabetic rats. Moreover, hepatic DNL‐regulated enzymes such as sterol regulatory element‐binding protein‐1 (SREBP1) and fatty acid synthase (FAS) expression were significantly suppressed by RTLE (100 and 400 mg/kg body weight) in diabetic rats. The evidences of this study suggest that RTL possesses potential on alleviating NAFLD and lipid accumulation via regulating DNL in the liver of HFD/STZ‐induced T2DM rats.


| Preparation of RTL extracts
The stems and leaves of RTL were purchased from the Herb Light farm, Yi-Lan County, Taiwan, in May of 2014 and identified by Prof. Wei-Jan Huang in the College of Pharmacy, Taipei Medical University. A voucher specimen (TMU27423) was deposited in the herbarium of College of Pharmacy, Taipei Medical University. All samples were washed, dried, weighed, sliced, and freeze dried.
Each 1 g dried stem or leaf was extracted with 6 ml of distilled water (RTLW) or 95% ethanol (RTLE) (1:6, w/v) individually at 4°C for 72 hr and then filtered through cheese cloth. The filtrate was further filtered twice through Whatman No. 1 filter paper before centrifuged at 4,700 × g for 20 min. The supernatant was vacuum concentrated using a rotary evaporator below 40°C. The concentrate was freeze dried into a powder and stored at −80°C until used.

| Animals and Diets
Male Wistar rats (age 4 weeks) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. The room conditions and treatment procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all of the protocols were approved by the Institutional Animal Care and Use Committee of National Taiwan Normal University, Taipei, Taiwan (approval number 103042). The rats were maintained under standard laboratory conditions at a temperature of 23 ± 1°C and a 12-hr light/12-hr dark cycle, with free access to food and water for the duration of the study.
HFD/STZ-induced T2DM rats were carried out by the method of Ma et al. (2014) with slight modifications. After 1-week adaptation, they were fed an HFD (60% calories from fat) for 4 weeks. STZ (28 and 15 mg/kg body weight, respectively, dissolved in 0.1 M sodium citrate buffer at pH 4.5) was intraperitoneally injected into rats at the 5th and 6th weeks to induce diabetes. The diabetic rats were then fed HFD for another 6 weeks prior to experimental procedures to guarantee the stable phenomena of hyperglycemia. For the animal experimental design, the rats were divided into seven groups (each contains six rats): Group 1 consists of rats fed a normal diet for 11 weeks; Group 2 diabetic rats fed an HFD (60% calories from fat) for 11 weeks as the negative control; Group 3 diabetic rats fed an HFD and orally administered pioglitazone (Pio; 30 mg/kg body weight) daily during the last 4 weeks of the 11 weeks' experiment as the positive control; Groups 4 and 5 diabetic rats fed an HFD and orally administered RTLW (100 or 400 mg/kg body weight, respectively) daily during the last 4 weeks of the 11 weeks' experiment; and Groups 6 and 7 diabetic rats fed an HFD and orally administered RTLE (100 or 400 mg/kg body weight, respectively) daily during the last 4 weeks of the 11 weeks' experiment. The body weight was monitored each week. The liver weight and adipose (epididymal and perirenal adipose tissues) weight were monitored after scarification at the end of the experiment. The livers were stored at −80°C for triglyceride determination and Western blot analysis.

| Hepatic triglyceride assay
Hepatic triglyceride assay was carried out by Triglyceride Colorimetric Assay Kit (Cayman Chemical, Co) and performed according to the protocol.

| Histopathological analysis
Liver tissue was removed and immediately fixed in 10% neutral phosphate-buffered formalin solution and embedded in paraffin. Sections 4-5 um thick were cut by a rotary microtome (Leica Microsystems, Wetzlar, Germany) and stained by hematoxylin-eosin (H & E). The stained specimens were observed and photographed by utilizing an upright digital imaging microscope (Zeiss Axioplan 2).

| Liver and epididymal adipose tissue protein preparation
The liver (0.5 g) or epididymal adipose (0.05g) was homogenized with lysis buffer (0.2% Triton X-100, 5 mmol/l EDTA, and 1 mmol/l phenylmethylsulfonyl fluoride) at 4°C for 2 min and then centrifuged (10,000 × g, 20 min, 4°C) to acquire the supernatant. The protein concentration in the cell extract was determined using a Bio-Rad protein assay.

| Western blot analysis
The Western blot was adopted by Huang, Wen-Chang Chang, Wu, Shih, and Shen (2016)

| Statistical analysis
Results are presented as the mean ± standard deviation (SD), which was analyzed statistically with SAS Version 9.4 (SAS Institute Inc, Cary, NC, USA) using one-way ANOVA and Duncan's new multiple range tests. All comparisons were made relative to the normal group, where p < .05 is considered to be statistically significant. Table 1 shows the changes of organ weight in rats after 4 weeks of administration of RTL extracts. Liver, perirenal, and epididymal adipose tissues from rats were acquired and weighed after sacrifice.

| Effect of RTL extracts on organ weight in HFD/ STZ-induced T2DM rats
HFD fed plus STZ injection rats (DM group) caused 23% and triple increase of relative liver and adipose tissue weights, respectively, in comparison to normal rats (p < .05; Table 1). Figure 1 showed that treatment of HFD and STZ caused 2.5 times increase in hepatic TG levels in type 2 diabetic rats (5.72 ± 1.04%), when compared with normal group (2.29 ± 0.20%) (p < .05). histochemical stain revealed that HFD and STZ treatment caused hypertrophy and tiny vacuoles on the inside of liver cell, also known as slight steatosis (Figure 2). RTL may significantly improve the progression of hypertrophy and tiny vacuoles.

| Effect of RTL extracts on the DNL-associated protein expression of fatty acid metabolism in liver of HFD/STZ-induced type 2 T2DM rats
In the present study, treatment of HFD and STZ caused 1.2 times increase in the expression of FAS as compared to that of N group (p < .05; Figure 3). The expression of FAS was decreased by 61.4%, 58.5%, and 49.5% in DM + PIO, DM + E100, and DM + E400 group, respectively, as compared with DM group after treatment of PIO or RTLE (Figure 3). The expression of hepatic SREBP1 was increased by 101.2% in DM group when compared with N group (p < .05; Figure 4). Enhanced expressions of hepatic SREBP1 were declined by 43.6%, 28.2%, 24.5%, 47.9%, and 43.6% in DM + PIO, DM + W100, DM + W400, DM + E100, and DM + E400 group, respectively, when compared with DM group (p < .05; Figure 4). Jensen, Caruso, Heiling, and Miles (1989)    lead to adipocyte hypertrophy, promote secretion of tumor necrosis factor-α and insulin-like growth factor, and consequently induce the growth of adipocytes. Pioglitazone was proved to promote adipocyte differentiation, reduce urine sugar, increase body fluid retention and appetite resulting in weight gain (Ghosh & Dey, 2011). Excessive fat intake may cause imbalance of lipid metabolism in liver, which also promote liver fat accumulation and tissue hypertrophy (Puigserver & Rodgers, 2006). Patients with F I G U R E 3 Protein expression of hepatic FAS in high-fat diet and streptozotocin-induced type 2 diabetic rats fed with Ruellia tuberosa L. (RTL) extracts for 4 weeks. Normal: Normal diet; DM: high-fat diet (HFD; 60% fat) plus STZ (28 mg/kg body weight, i.p.) induced diabetic rats; DM + Pio: DM rats gavaged with pioglitazone (30 mg/kg body weight) for 4 weeks; DM + W100: DM rats gavaged with RTL water extract (100 mg/kg body weight) for 4 weeks; DM + W400: DM rats gavaged with RTL water extract (400 mg/kg body weight) for 4 weeks; DM + E100: DM rats gavaged with RTL ethanol extract (100 mg/kg body weight) for 4 weeks; DM + E400: DM rats gavaged with RTL ethanol extract (400 mg/kg body weight) for 4 weeks. Values were calculated as the mean ± SD, n = 6 for each group. Notes: a-c letters = significant differences among all samples tested (p < .05)

F I G U R E 4
Protein expression of hepatic SREBP1 in high-fat diet and streptozotocin-induced type 2 diabetic rats fed with Ruellia tuberosa L. (RTL) extracts for 4 weeks. Normal: Normal diet; DM: high-fat diet (HFD; 60% fat) plus STZ (28 mg/kg body weight, i.p.) induced diabetic rats; DM + Pio: DM rats gavaged with pioglitazone (30 mg/kg body weight) for 4 weeks; DM + W100: DM rats gavaged with RTL water extract (100 mg/kg body weight) for 4 weeks; DM + W400: DM rats gavaged with RTL water extract (400 mg/kg body weight) for 4 weeks; DM + E100: DM rats gavaged with RTL ethanol extract (100 mg/kg body weight) for 4 weeks; DM + E400: DM rats gavaged with RTL ethanol extract (400 mg/kg body weight) for 4 weeks. Values were calculated as the mean ± SD, n = 6 for each group. Notes: a-b letters = significant differences among all samples tested (p < .05) NAFLD usually accompany the progression of hepatomegaly (Zeng et al., 2008). Results of this study revealed that RTLW and RTLE significantly reduced relative liver and adipose tissue weights in HFD/STZ-induced diabetic rats.

Dietary fat is decomposed by lipoprotein lipase in intestine and
transported to adipose tissue for storage. TG in adipose tissue is degraded into glycerol and FFA by hormone-sensitivity lipase, resulting in elevated FFA level. FFA was integrated with serum albumin and then transported to liver to stimulate the secretion of VLDL (Zechner, Strauss, Haemmerle, Lass, & Zimmermann, 2005).
Insulin resistance has been proved to cause abnormal regulation of lipolysis in peripheral adipocytes, leading to massive release of FFA into blood (Petersen & Shulman, 2006). Our previous study has revealed that RTLW and RTLE may alleviate hyperglycemia and hyperlipidemia via improving insulin resistance in HFD/STZinduced type 2 diabetic rats Ko et al., 2019). In the normal condition, hepatic triglyceride of less than 5% is derived from endogenous lipid synthesis in the liver. Excessive fat intake from high-fat diet may deposit in adipose tissue and organs as the form of TG, resulting in NAFLD (Ji, Zhao, Leng, Liu, & Jiang, 2011) when fat accumulation is high than 5% in liver tissue (Loomba & Sanya, 2013). Fat accumulation in liver was also considered an indicator of hepatic insulin resistance (Kotronen & Yki-Järvinen, 2008).
Obese insulin resistance may increase the risk for abnormal accumulation of fat through induction of lipid metabolism disorder in liver (Loomba & Sanya, 2013 (Abraham et al., 2016). SREBP1 was reported to positive regulated FAS expression, which caused TG synthesis (Goedekeet al., 2018). HFD has been proved to cause TG accumulation via increasing SREBP-1c and FAS expression (Ji et al., 2011).
This study could be comprehended that RTLW decreased FAS expressions via suppressing the expression of SREBP1 to reduce TG production in liver and subsequently ameliorate NAFLD in HFD/ STZ-induced T2DM rats.

| CON CLUS IONS
The present study first demonstrated that RTLW may decrease DNL via downregulating SREBP1 and FAS expressions in liver, while inhibiting the accumulation of hepatic TG and formation of NAFLD in HFD/STZ-induced diabetic rats. We suggested that RTL may be a potential therapy for amelioration on hepatic dyslipidemia and steatosis in T2DM rats. The purification and identification of active components in RTL extracts are currently on the way for further investigation in our laboratory.

ACK N OWLED G M ENTS
The authors would like to thank the Ministry of Science and

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 S S TATEM ENT
The study was conducted in accordance with the ethical guidelines of the Institutional Animal Care and Use Committee of National Taiwan Normal University, Taipei, Taiwan (approval no. 103042).