Prevention and treatment effect of total flavonoids in Stellera chamaejasme L. on nonalcoholic fatty liver in rats

The feeding of high-fat diet for 4 weeks effectively induced NAFLD in rats, as evidenced
by the markedly increased body weight, liver weight, and serum TG, TC, LDL-C, ALT,
AST and TBA, while decreasing HDL-C (Table 2). Furthermore, HE staining results (Fig. 1(b)) showed that steatosis was developed and numerous lipid droplets were observed in
all of the five livers from the NAFLD rats. This high-fat-diet-induced NAFLD model
has the same key pathological features as those reported for other rat NAFLD models
3], 17], 18]. Based on the previous results, the NAFLD model was considered to be successfully
established.

Table 2. Effect of total flavonoids in Stellera chamaejasme L. on weight (g) of rats (mean?±?SD) (n?=?20)

Fig. 1. HE staining for histological evaluation. Typical photographs of liver sections of
normal rat (a) and NAFLD rat (b) in 4 weeks; (c)normal rat (negative control group); (d) the rat treated with TFSC (100 mg/kg) for 6 weeks; (e) the rat treated with TFSC (200 mg/kg) for 6 weeks; (f) the rat treated with TFSC (400 mg/kg) for 6 weeks; (g) positive control group. (Magnification 400x)

In the following 6 weeks, rats of high-dose group and middle-dose group showed significantly
reduced body weight (by 5.89 % and 4.10 % respectively (P??0.05 for both), liver weight (by 24.99 %, P??0.01 and 17.87 %, P? 0.05 respectively), and liver index (liver/body weight, by 21.02 %, P??0.01 and 15.10 %, P??0.05 respectively) compared with rats with the positive control group (NAFLD rats).
No statistical difference was found between Low-dose group and the positive control
group (Table 2).

Results of AST, ALT, TG, TC, LDL-C, HDL-C, GLU,TBA, Leptin, concentration of rats
administered different doses of TFSC are shown in Tables 3. Rats administered 400 mg/kg body weight of TFSC had significant (P??0.01) decrease in TG, TC and MDA compared with the positive control group, while
the groups administered 200 mg/kg and 100 mg/kg did not differ significantly in TG
and TC, but had decreased and MDA compared with the positive control group (P??0.05). Rats administered 400 mg/kg body weight of TFSC had significant (P??0.05) decrease in AST, ALT, LDL-C and TBA compared with the positive control group,
while the groups administered 200 mg/kg and 100 mg/kg did not differ significantly
in AST, ALT, LDL-C, but had decreased TBA compared with the positive control group
(P??0.05). Rats administered 400 mg/kg body weight of TFSC had significant (P??0.05) increase in HDL-C compared with the positive control group, while the groups
administered 200 mg/kg and 100 mg/kg did not differ significantly in HDL-C compared
with the positive control group (P??0.05). Meanwhile, hepatic histopathological examination showed the steatosis area
and the number of lipid droplets were decreased in the different doses of TFSC-treated
group, indicating the improvement of liver steatosis (Fig. 1d, e, f).

Table 3. Effect of total flavonoids in Stellera chamaejasme L. on serum lipid level in rats (mean?±?SD) (n?=?20)

The results showed that TFSC significantly increased in T-SOD, GSH-Px activity and
reduced in MDA level both in rat serum.

After stained with HE, the degrees of hepatic steatosis were examined. While there
was no obvious steatosis in the liver of the normal group of rats, different degrees
of liver steatosis were observed in other group rats (Figs. 1 and 2). The area of hepatic steatosis in the high-dose group decreased remarkably when
compared with that in the positive control group (P??0.01), in the middle-dose group decreased when compared with that in the positive
control group (P??0.05), the score of hepatic steatosis in the low-dose group decreased when compared
with that in the positive control group (P??0.05), however, they remained significantly higher than that of the high-dose group
or middle-dose group. There was no significant difference in the score of liver steatosis
between the high-dose group and middle-dose group (P??0.05) (Figs. 1 and 2).

Fig. 2. Scores of hepatic steatosis of rat livers. The scores were determined, according to
the the percentage of hepatocytes containing lipid droplets. Data are expressed as
mean?±?SD of each group (n?=?20 per group) and determined by a pathologist in a blinded fashion. *P??0.05, **P??0.01, compared to normal control group, # P??0.05, ## P??0.01, compared to positive control group

We next examined whether the different doses of TFSC supplementation affected mRNA
expression of genes involved in hepatic lipid metabolism in liver samples of rats.
The mRNA levels of genes such as PPAR-?, SREBP-1c, ACC, FAS and LEP showed a 53 %,
35 %, 47 %, 48 % and 43 % decrease, respectively, in the high-dose group compared
with those of the positive control group (NAFLD rats) (Fig. 3a). the mRNA levels of PPAR-?, SREBP-1c, ACC, FAS and LEP showed a 15 %, 26 %, 19 %,
19 % and 22 % decrease, respectively, in middle-dose group, compared with those of
the positive control group (NAFLD rats) (Fig. 3a). Also, the mRNA levels of PPAR-?, SREBP-1c, ACC, FAS and LEP showed a 6 %, 7 %,
14 %, 16 % and 13 % decrease, respectively, in the low-dose group, compared with those
of the positive control group (NAFLD rats) (Fig. 3a). The mRNA levels of PPAR-?, CPT-1, and CYP7A1 showed 3.26-, 3.35-, and 2.56-fold
increase, respectively, in the high-dose group compared to the positive control group
(Fig. 3b). The mRNA levels of PPAR-?, CPT-1, and CYP7A1 showed 2.01-, 1.58-, and 1.85-fold
increase, respectively, in the middle -dose group compared to the positive control
group (Fig. 3b). The mRNA levels of PPAR-?, CPT-1, and CYP7A1 showed 1.27-, 1.21-, and 1.38-fold
increase, respectively, in the low-dose group compared to the positive control group
(Fig. 3b).

Fig. 3. The expression of lipid metabolic regulators in the liver. (A) The expression of hepatic PPAR-?and related genes. The relative expression levels
of hepatic PPAR-?, ACC, SREBP-1c, LEP and FAS mRNA transcripts were determinedby real-time
PCR in liver samples of rats from the positive control group (NAFLD rats) and the
different doses of TFSC-treated group (n =20 per group). (B) The expression of CYP7A1and related genes. The relative expression levels of hepatic
CYP7A1, CPT-1 and PPAR-? mRNA transcripts were determined by real-time PCR in liver
samples of rats from the positive control group (NAFLD rats) and the different doses
of TFSC-treated group (n =20 per group). Values are means ± positive and negative
errors. Differences between groups were determined by Student’s t test. *P0.05; **P0.01 versus the positive control group (NAFLD rats). Abbreviations: CYP7A1, Cholesterol
7?-hydroxylase1; CPT, carnitine palmitoyltransferase; PPAR, peroxisome proliferator-activated
receptor; SREBP, sterol regulatory element-binding protein; ACC, Acetyl-CoA carboxylase,
FAS, Fatty acid synthase, LEP, Leptin

Our studies indicated that TFSC had lowering lipid and hepatoprotective effect, and
regulated and improved the balancing effects of free radicals on rat model of NAFLD.
It’s possible mechanism is that TFSC downregulating the mRNA expression of transcription
factor SREBP-lc to regulate the downstream lipid synthesis related genes ACC, FAS
and LEP expression, meanwhile, TFSC upregulated the level of transcription factor
PPAR-? to regulate the downstream lipidolysis related genes CPT-1 and CYP7A1 expression,
and improved the liver tissue ? oxidation and in vivo fatty acid degradation, inhibit
fatty acid synthesis, and finally reach the effective regulation of lipid metabolisms
of experimental rat model of NAFLD (Table 4).

Table 4. Effects of Total Flavonoids in Stellera chamaejasme L. on liver lipid level in rats (mean?±?SD) (n?=?20)