An Integrative Pharmacology Based Analysis of Refined Liuweiwuling Against Liver Injury: A Novel Component Combination and Hepaprotective Mechanism

Abstract

Liver disease is a major cause of illness and death worldwide. In China, liver diseases, primarily alcoholic and nonalcoholic fatty liver disease, and viral hepatitis, affect approximately 300 million people, resulting in a major impact on the global burden of liver diseases. The use of Liuweiwuling (LWWL), a traditional Chinese medicine formula, approved by the Chinese Food and Drug Administration for decreasing aminotransferase levels induced by different liver diseases. Our previous study indicated a part of the material basis and mechanisms of LWWL in the treatment of hepatic fibrosis. However, knowledge of the materials and molecular mechanisms of LWWL in the treatment of liver diseases remains limited. Using pharmacokinetic and network pharmacology methods, this study demonstrated that the active components of LWWL were involved in the treatment mechanism against liver diseases and exerted anti-apoptosis and anti-inflammatory effects. Furthermore, esculetin, luteolin, schisandrin A and schisandrin B may play an important role by exerting anti-inflammatory and hepatoprotective effects in vitro. Esculeti and luteolin dose-dependently inhibited H₂O₂-induced cell apoptosis, and luteolin also inhibited the NF-κB signaling pathway in bone marrow-derived macrophages. schisandrin A and B inhibited the release of ROS in acetaminophen (APAP)-induced acute liver injury in vitro. Moreover, LWWL active ingredients protect against APAP-induced acute liver injury in mice. The four active ingredients may inhibit oxidative stress or inflammation to exert hepatoprotective effect. In conclusion, our results showed that the novel component combination of LWWL can protect against APAP-induced acute liver injury by inhibiting cell apoptosis and exerting anti-inflammatory effects.

Keywords: anti-inflammation; antiapoptosis; component combination; hepatoprotective effect; liuweiwuling; network pharmacology.

FIGURE 1

Prediction of the effective targets of LWWL with network pharmacology.

FIGURE 2

Compound–target interaction network and preliminary gene ontology (GO) analysis of drug targets. (A) the potential targets of LWWL mainly distributed in the cellular component, (B) the potential targets of LWWL can bind and activate molecular function, (C) the potential targets engaged in the biological process, and (D) KEGG pathway analysis of LWWL.

FIGURE 3

Effects of the active components of LWWL on cell viability. The viability of L02 cells treated with the active components of LWWL for 24 h was determined. Data are presented as mean ± SD using biological samples.

FIGURE 4

Luteolin inhibits the NF-κB signaling pathway. (A) Western blotting of pro IL-1β, NLRP3, Casp-1 p45, ASC, and GAPDH in BMDMs treated with apigenin, esculetin, gomisin N, schisanhenol, schisandrin A, schisandrin B, anwulignan, schisantherin A, schisandrin B, specnuezhenide, schisandrin, luteolin, quinic acid, and curcumenol (40 μM) for 4 h and then stimulated with LPS (50 ng/ml) for 1 h. (B,C) ELISA of TNF-α (B) and IL-6 (C) in SN from samples described in A. (D) Western blotting of pro IL-1β, NLRP3, Casp-1 p45, ASC, and GAPDH in BMDMs treated with luteolin for 4 h and then stimulated with LPS (50 ng/ml) for 1 h. (E,F) ELISA of TNF-α (E) and IL-6 (F) in SN from samples described in D. GAPDH served as a loading control. Data are represented as the mean ± SD using biological samples.

FIGURE 5

Luteolin and esculetin suppress H₂O₂-induced apoptosis. (A) Apoptosis of L02 cells treated with apigenin, esculetin, gomisin N, schisanhenol, schisandrin A, schisandrin B, anwulignan, schisantherin A, schisantherin B, specnuezhenide, schisandrin, luteolin, quinic acid, and curcumenol (40 μM) and then exposed to APAP, as detected by flow cytometry. (B) The percentage of early apoptotic cells from samples described in A. (C) The percentage of total apoptotic cells from samples described in A. (D) Apoptosis of L02 cells treated with esculetin or luteolin (5, 10, and 20 μM) and then exposed to APAP, as detected by flow cytometry. (E–G) The percentage of early apoptotic cells (E), late apoptotic cells (F), and total apoptotic cells (G) treated with luteolin (5, 10, and 20 μM). (H–J) The percentage of early apoptotic cells (H), late apoptotic cells (I), and total apoptotic cells (J) treated with esculetin (5, 10, and 20 μM). Data are represented as the mean ± SD using biological samples. The significance of the differences was analyzed using unpaired Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001 vs. the control, NS, not significant.

FIGURE 6

Schisandrin A and schisandrin B inhibit the release of ROS. (A) HepaG2 cells were treated with apigenin, esculetin, gomisin N, schisanhenol, schisandrin A, schisandrin B, anwulignan, schisantherin A, schisantherin B, specnuezhenide, schisandrin, luteolin, quinic acid, and curcumenol (40 μM) before being stimulated with APAP. HepaG2 were loaded with MitoSOX red mitochondrial superoxide indicator (Ex/Em: 510/580 nm). After staining and washing, flow cytometry was conducted to test mtROS production. (B) Percentage of ROS-positive cells in HepaG2 cells from samples described in A. (C) The production of mtROS was detected by flow cytometry in HepaG2 cells treated with schisandrin A or schisandrin B (10, 20, and 40 μM). (D,E) Percentage of ROS-positive cells in HepaG2 cells pretreated with schisandrin A (D) or schisandrin B (E) (10, 20, and 40 μM) and then stimulated with APAP, followed by staining with MitoSox. Data are represented as the mean ± SD using biological samples. The significance of the differences was analyzed using unpaired Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001 vs. the control, NS, not significant.

FIGURE 7

Combination of LWWL active ingredients protect against APAP-induced acute liver injury in vivo. (A–G) Eight-week-old C57BL/6 male mice were administered with a vehicle, schisandrin A, schisandrin B, esculetin, and luteolin every day by gavage for 7 days. Further, 1 h after the final gavage of schisandrin A, schisandrin B, esculetin and luteolin, mice in all groups (except the control group) were administered with APAP (300 mg/kg) by a single intraperitoneal injection. (A–D) Serum levels of ALT(A), AST(B), DBIL (C) and TBA (D). (E,F) ELISA of IL-1β (E) and TNF-α (F). (G) H&E staining. The significance of the differences was analyzed using unpaired Student’s t-test: #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. the APAP group, NS, not significant.