Quantitative analysis in FZHY decoction and FZHY-biological samples
The chemical structure and concentration of 16 compounds in FZHY decoction (2 g/mL) were displayed in Fig.S1 and Table S3, respectively. The concentration–time curves of compounds in the plasma and liver after oral administration of FZHY decoction (20 g/kg) in rats was shown in Fig. 1a and 1b, and the corresponding PK parameters were shown in Table 1. The summary of the contents and AUCs of compounds in the FZHY decoction or FZHY- biological samples were shown in Fig. 1c.
Table 1
The PK parameters of 11 compounds in the portal vein plasma, systemic plasma and liver, following oral administration of FZHY decoction in rats.
Compounds | Portal vein plasma | Systemic plasma |
Tmax(h) | Cmax(ng/ml) | AUC(h*ng/ml) | Tmax(h) | Cmax(ng/ml) | AUC(h*ng/ml) |
Salvianolic acid A | 0.167 | 26.7 ± 11.2 | 108.7 ± 9.1 | 0.167 | 33.1 ± 14.9 | 122.3 ± 5.7 |
Salvianolic acid B | 0.167 | 74.8 ± 36.4 | 144.4 ± 7.3 | 0.167 | 61.5 ± 42.1 | 55.9 ± 18.6 |
Danshensu | 0.167 | 279.8 ± 58.8 | 1515.9 ± 64.6 | 0.5 | 206.6 ± 41.6 | 1362 ± 95 |
Gypenoside XLIX | 0.5 | 21.5 ± 7.3 | 21.3 ± 3.5 | 0.5 | 22.4 ± 9.8 | 26.4 ± 3.8 |
Amygdain | 0.167 | 347.7 ± 82.5 | 254.7 ± 23.2 | 0.167 | 286.2 ± 44.2 | 262.8 ± 23.9 |
Rosmarinic acid | 0.167 | 27.1 ± 8.1 | 51.9 ± 4 | 0.167 | 15.4 ± 4.9 | 45.5 ± 4.7 |
Schisandrol A | 0.167 | 68.2 ± 7.2 | 62 ± 8.6 | | | |
Schisandrol B | 0.5 | 43.4 ± 3 | 215.1 ± 49.3 | 0.5 | 35.9 ± 0.6 | 84.3 ± 19.8 |
Deoxyschizandrin | 0.167 | 4.4 ± 1.8 | 15.6 ± 1.1 | | | |
Schisandrin B | 0.5 | 29.9 ± 11 | 89.4 ± 15.7 | | | |
Schisantherin A | 0.5 | 1657.7 ± 584.3 | 8542.1 ± 566.2 | 0.5 | 2094.4 ± 681 | 8672.6 ± 1093.6 |
| Liver | | | |
Compouds | Tmax(h) | Cmax(ng/g) | AUC(h*ng/g) | | | |
Amygdain | 7 | 185 ± 24.1 | 4423.2 ± 599.8 | | | |
Schisandrol A | 0.5 | 42.5 ± 8.4 | 96.4 ± 13.7 | | | |
Schisandrol B | 14 | 29.9 ± 1.8 | 578.7 ± 50.3 | | | |
Schisandrin B | 0.5 | 18.7 ± 1.7 | 24.1 ± 4.6 | | | |
Schisantherin A | 0.5 | 1011.1 ± 322.6 | 1070.3 ± 103.4 | | | |
There were 16 compounds determined in the FZHY decoction, including 6 compounds derived from Salvia miltiorrhiza, 5 compounds derived from Schisandra chinensis, 2 compounds derived from Gynostemma pentaphylla, 2 compounds from Cordyceps mycelium and 1 compound from Gynostemma pentaphylla. Among the compounds, salvianolic acid B was of the highest content in FZHY decoction, followed by danshensu, amygdalin, schisantherin A and salvianolic acid A, as the members of the high content group (concentration ≥ 1000 µg/ml). Adenosine, gypenoside XLIX, rosmarinic acid, and schisandrol A were subjected to the middle content group (100 µg/mL༜concentration༜1000 µg/mL), and the concentrations of the rest compounds, including schisandrol B, deoxyschizandrin, ginsenoside Rb3, schisandrin B, tanshinone I, cryptotanshinone and cordycepin, were very low(concentration ≤ 100µ g/ml)
Portal vein blood is the first site after gut absorption but before hepatic disposition, which is responsible for transferring the substances to the liver post-dose. Following oral administration of FZHY in rats, there were 11 compounds accurately detected in the portal vein plasma, where tanshinone I, cryptotanshinone, cordycepin, ginsenoside Rb3 and adenosine were undetected, probably due to the low content in the formula or the poor physicochemical property. The Tmaxs of all the compounds were within 0.5 h in portal vein plasma, indicating of the fast absorption. Schisantherin A exhibited the maximum exposure in the portal vein plasma, followed by danshensu (AUC ≥ 1000h*ng/mL). There were 4 compounds,including amygdalin༌schisandrol B, salvianolic acid B, and salvianolic acid A belonged to the middle exposure group (100 h*ng/mL༜concentration༜1000 h*ng/ml).The rest 5 compounds were subjected to the low exposure group (AUC ≤ 100 h*ng/mL).
After hepatic disposition, the compounds were transported to the systemic plasma, which is responsible for delivering the substances to the other organs, except liver. Compared to those in the portal vein plasma, there were 8 compounds determined in the systemic plasma, where schisandrol A, schisandrin B, and deoxyschizandrin were undetected. Similar to those in the portal vein plasma, the absorption of those compounds was quick, and schisantherin A and danshensu were attributed to the high exposure group (AUC ≥ 1000 h*ng/mL), and schisantherin A were of the highest exposure in the systemic plasma. The middle exposure group (100 h*ng/mL༜AUC༜1000 h*ng/ml) included of amygdalin and salvianolic acid A, and the rest compounds belonged to the low exposure group (AUC ≤ 100 h*ng/ml).
Following oral administration of FZHY decoction, there were only 5 compounds detected in the liver. The Tmaxs of schisandrol A, schisandrin B and schisantherin A were at 0.5 h, similar to those in the plasma. In contrast, those of schisandrol B and amygdalin were at 14 h and 7 h, respectively, consistent to their multi-peak phenomenon in the concentration-time curves. Contrary to those in the plasma, amygdalin exhibited the highest hepatic exposure, followed by schisantherin A, whose exposure were more than 1000 h*ng/g. Schisantherin A belong to the middle exposure group (100 h*ng/g༜AUC༜1000 h*ng/g), and the hepatic exposure of schisandrol A and schisandrin B was very low (AUC ≤ 100 h*ng/g). Finally, salvianolic acid B, schisantherin A and amygdalin, who were of the maximum quantity in the FZHY decoction, plasma and liver, respectively, were selected to combined to evaluate the anti-hepatic fibrosis effect in vivo.
JY5 significantly alleviate hepatic injury and collagen deposition in CCl 4 -induced rat and mouse liver fibrosis
Compared with the control group, the levels of serum ALT and AST were significantly increased in the CCl4 group. After treatment with JY5 or FZHY, the levels of ALT and AST were significantly decreased (Fig. 2a, 2b and Fig. S2d, S2e). The serum AST level was decreased in the SORA group compared with the CCl4 group (Fig. 2b).
H&E staining showed that the hepatic lobular structure was severely collapsed with more complete pseudo-lobules formed in the CCl4 group. And fibrous tissue became denser, and hepatocytes were disordered and ballooning degeneration. There were a large number of inflammatory cells infiltration surrounding the hepatic sinusoid, central vein and portal tract. The above lesions were obviously attenuated with less pseudo-lobules and inflammatory cells infiltration, after treatment with JY5 or FZHY or SORA (Fig. S2a and Fig. 2c, upper panel).
SR staining showed that compared to the control group, the collagen deposition was obviously increased in the CCl4 group. The fibrotic septum became significantly widened and distributed from the portal tract to the periphery in a reticular manner, forming pseudo-lobules with varying sizes. In contrast, the collagen deposition was obviously decreased, the fibrotic septum became narrower, and pseudo-lobules structures were observed less in the JY5 or FZHY or SORA treated groups (Fig. S2a and Fig. 2c, middle panel). Both the hepatic Hyp content and collagen deposition were significantly increased in the CCl4 group, compared to the control group. The above indicators were significantly reduced after the intervention with JY5 or FZHY or SORA (Fig. S2b, S2c and Fig. 2d, 2e). These results demonstrated that JY5 formula had a significantly anti-liver fibrosis comparably to that of FZHY.
IHC staining showed that compared with the control group, numerous Col-Ⅰ expression were visible in the fibrotic septum in the CCl4 group. By contrast, JY5 and SORA could significantly reduce Col-Ⅰ expression in the liver tissue (Fig. 2c, lower panel). In addition, qRT-PCR results showed that Col-Ⅰ mRNA expression was significantly more elevated in the CCl4 group than that in the control group. Whereas compared to the CCl4 group, Col-Ⅰ mRNA expression was significantly reduced in JY5 treated group (Fig. 2f).
Consistent with the CCl4-induced rat liver fibrosis model, JY5 could significantly alleviate hepatic injury and collagen deposition in CCl4-induced liver fibrosis in mice (Fig. 3).
JY5 significantly alleviate hepatic injury and collagen deposition in BDL-induced rat liver fibrosis
Compared with the sham group, the levels of ALT, AST, TBil, DBil, TBA and ALP were significantly increased in the BDL group. After JY5 or DAPT treatment, the levels of ALT, AST, TBil, DBil, TBA and ALP were significantly decreased (Fig. 4a-4f).
Consistent with the CCl4-induced liver fibrosis, JY5 could reduce the hepatic Hyp content and collagen deposition, meanwhile down-regulate the expressions of Col-Ⅰ and Col-IV in BDL-induced rat liver fibrosis (Fig. 4g-4i). These results suggested that JY5 could significantly alleviate hepatic injury and collagen deposition in BDL-induced liver fibrosis in rats.
JY5 significantly represses the activation of HSCs in vivo
Both in the CCl4-induced rat and mouse liver fibrosis experiments, IHC staining showed that numerous α-SMA and Desmin expression were visible in the fibrotic septum in the CCl4 group. By contrast, both α-SMA(+) cells and Desmin(+) cells were decreased in the JY5 and SORA treated group (Fig. 5a, 5d). Western blot and qRT-PCR showed that the α-SMA expression was significantly elevated compared to the control group in the CCl4 group. Whereas compared to the CCl4 group, both α-SMA protein and mRNA expressions were significantly reduced in JY5 and SORA treated groups (Fig. 5b, 5c, 5e and 5f). Similarly, in the BDL-induced liver fibrosis experiment, the treatment effect of JY5 was consistent with these results in the CCl4-induced liver fibrosis experiments (Fig. 5g-5i). These results demonstrated that JY5 significantly represses the activation of HSCs in CCl4 -and BDL-induced liver fibrosis.
JY5 significantly inhibit the activation of Notch signaling pathway in vivo
In the CCl4-induced liver fibrosis rat experiment, qRT-PCR showed that the mRNA expressions of Notch2, Notch3, Notch4, Jagged1, Jagged2 and recombination signal binding protein-κB (RPB-κB) were significantly more up-regulated in the CCl4 group than these genes in the control group. Whereas compared to the CCl4 group, Notch2, Notch3, Notch4, Jagged1, Jagged2 and RPB-κB mRNA expressions were significantly reduced in JY5 and SORA treated groups (Fig. 6a). Western blot showed that the protein expression of RPB-κB was significantly increased compared to the control group in the CCl4 group. RPB-κB protein expression was significantly more reduced in JY5 and SORA treated groups than that in the CCl4 group (Fig. 6b). While in the CCl4-induced liver fibrosis mice experiment, JY5 could not only decrease the expressions of Notch2, Notch3, Notch4, Jagged1 and RPB-κB, but also down-regulate the expression of Dll1 (Fig. 6c and 6d). Consistent with the CCl4-induced rat liver fibrosis model, JY5 could decrease the expressions of Notch2, Notch3, Notch4, Jagged1 and RPB-κB in the BDL-induced liver fibrosis model. In addition, the expressions of Dll1, Dll4 and Jagged 2 were significantly decreased after treatment with JY5 (Fig. 6e and 6f). These results suggested that JY5 could significantly inhibit the activation of Notch signaling pathway in CCl4 -and BDL-induced liver fibrosis.
JY5 might inhibit activation of LX-2 cells induced by TGF-β1 via regulating Notch signaling pathway
LX-2 cells were activated by TGF-β1 to observe the effect of JY5 at various concentrations in vitro. qRT-PCR showed that the mRNA levels of α-SMA, Col-Ⅰ, Notch3 and Jagged1 were significantly elevated in the TGF-β1 treated cells compared to the control cells. Whereas a-SMA、Col-Ⅰ、Notch3 and Jagged1 mRNA expressions were significantly reduced after treatment with various concentrations of JY5. And Col-Ⅰ、Notch3 and Jagged1 mRNA expressions were significantly decreased in the high-dose of JY5 treated group, compared to the low-dose of JY5 treated group (Fig. 7b-7e). Western blot results showed that the protein expressions of a-SMA and RBP-κB were significantly increased after treated with TGF-β1. Compared to the TGF-β1 group, both a-SMA and RBP-κB protein expressions were significantly reduced in various concentrations of JY5 treated groups. Among of these, the protein expression of RBP-κB was significantly reduced in the high-dose of JY5 treated group compared to the middle-dose and low-dose of JY5 treated groups (Fig. 7a and 7f). The above results suggested that JY5 might inhibit the activation of LX-2 cells induced by TGF-β1 via regulating Notch signaling pathway.