## 3 Results
### Effect of Orientin on Hepatic Fibrotic Signaling Markers ZEB-2, PTEN, and TNF-\(\alpha\)  
Orientin regulated the fibrotic markers in HSCs. The mRNA and protein expression of ZEB-2, PTEN, and TNF-\(\alpha\) were quantified in orientin-treated LPS-challenged HSCs (Figure 7). The ZEB-2 mRNA was downregulated from 4.4-fold of the LPS-induced condition to 3.1-fold of the orientin-treated condition (Figure 7A). Similar findings were observed in protein expression of ZEB-2 and reduced from 6.2-fold to 2.4-fold (Figure 7B,C). This indicates orientin controlled the mesenchymal modification of HSCs and inhibited the hepatic inflammation. HSC activator PTEN was inhibited by orientin treatment. The mRNA and protein expression of PTEN was significantly downregulated in orientin-treated HSCs.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Binding** & **Ligand** & **Intermole** & **Ligand Atoms** & **Docked Amino Acid Residue** \\
**Energy** & **Efficiency** & **Energy** & **(Ring)** & **(Bond Length)** \\ \hline \hline \multirow{7}{*}{\(-6.44\)} & \multirow{7}{*}{\(-0.2\)} & \multirow{7}{*}{\(-6.44\)} & \multirow{7}{*}{\(-6.44\)} & \multirow{7}{*}{\(-6.44\)} & \multirow{7}{*}{\(-6.44\)} \\  &Orientin inhibited the LPS-induced PTEN 3.4-fold of mRNA and 2.5-fold of protein expression into 2.6-fold and 1.9-fold of mRNA and protein, respectively (Figure 7A-C). The inflammatory marker TNF-\(\alpha\) was significantly inhibited by orientin treatment and it was downregulated from 4.9-fold of LPS-induced mRNA to 3.1-fold of mRNA. Orientin treatment of LPS-treated HSC led to a decrease of TNF\(\alpha\) protein levels (2.8-fold), when compared to HSCs treated only with LPS (4.5-fold) (Figure 7A-C). The ZEB-2 expression was increased in the orientin alone treated group compared to the untreated control group (Figure 7B,C).  
Figure 4: In silico docking of orientin (CID: 5281675) and (**A**) mouse PTEN (PDB-ID-1D5R) and (**B**) ZEB-2 (PDB-ID-2D8V) proteins binding analysis. Computational binding was performed using AutoDock software to demonstrate the illustration of interactions in the hydrophobic bond and other polar bonds of PTEN and ZEB-2. It shows the amino acid residue analysis of the interacted bond and its length, together with the binding pocket of ligand–receptor interactions.  
Figure 5: Effect of orientin on LPS-induced inflammatory HSCs. Naive mouse-derived HSCs were isolated and cultured in an adherent-coated 12-well plate. Cultured cells were stimulated with LPS 10 ng for 2 h and orientin was treated for 12 h. Treated cells were evaluated for cell proliferation, nitrate quantification, and iNOS protein. (**A**) Cell proliferation of LPS-stimulated HSCs and LPS with orientin-treated cells were quantified using SRB assay. (**B**) Nitric oxide level as nitrate (uM) in LPS-stimulated and orientin-treated HSCs. (**C**,**D**) iNOS protein quantification using immunoblot. Values are expressed as three independent experiments and all values are expressed as mean \(\pm\) SD. \({}^{\star}\) Represents the significant comparison with control and LPS-induced groups.  
Figure 6: Effect of orientin on LPS-induced inflammatory HSCs. Naive mouse-derived HSCs were isolated and cultured in an adherent-coated 12-well plate. Cultured cells were stimulated with LPS 10 ng for 2 h and treated with orientin (30 uM) for 12 h. (**A**) Control group, (**B**) LPS-treated group, (**C**) orientin (30 uM)-treated group, and (**D**) LPS and orientin (30 uM)-treated group.