Polysaccharides Extracted From Panax Ginseng C.A. Mey Enhance Complement Component 4 Biosynthesis in Human Hepatocytes

Abstract

Panax ginseng C.A. Mey (ginseng) is a classic medicinal plant which is well known for enhancing immune capacity. Polysaccharides are one of the main active components of ginseng. We isolated water-soluble ginseng polysaccharides (WGP) and analyzed the physicochemical properties of WGP including molecular weight, monosaccharide composition, and structural characteristics. WGP had minimal effect on the growth of hepatocytes. Interestingly, WGP significantly increased the mRNA and protein levels of complement component 4 (C4), one of the core components of the complement system. Promoter reporter gene assays revealed that WGP significantly enhanced activity of the C4 gene promoter. Deletion analyses determined that the E-box1 and Sp1 regions play key roles in WGP-induced C4 transcription. Taken together, our results suggest that WGP promotes C4 biosynthesis through upregulation of transcription. These results provide new explanation for the intrinsic mechanism by which ginseng boosts human immune capacity.

Keywords: C4 promoter; C4 transcription; complement component 4; ginseng; water-soluble ginseng polysaccharides.

FIGURE 1

Chromatographic analysis of WGP. HPGPC was used to determine WGP molecular weight distribution (A). HPLC was used to separate monosaccharide standards (B) and analysis the monosaccharides composition in WGP (C). Man, GlcA, Rha, GalA, Glc, Gal, Xyl, Ara, and Fuc represent mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose, respectively.

FIGURE 2

Structural characterization of WGP. FT-IR spectrum with the range of 4,000–400 cm⁻¹ (A) and ¹H NMR (B), ¹³C NMR (C) spectra were used to characterize WGP structure.

FIGURE 3

WGP have minimal effect on the growth of hepatocytes. L-O2 cells were treated with WGP at a series of equal ratio gradient concentrations for 72 h and then subjected to MTT assay. Data are presented as mean ± standard errors of the mean (SEM) from 3 independent experiments.

FIGURE 4

WGP increase C4 production in hepatocytes. L-O2 cells were treated with WGP at a series of equal ratio gradient concentrations for 24, 48 or 72 h. Protein levels of C4 were determined using western blotting. The fold changes of C4 densitometry measurements were compared to β-actin and then normalized to the vehicle control.

FIGURE 5

WGP promote C4 gene transcription. L-O2 cells were treated with WGP at a series of equal ratio gradient concentrations for up to 72 h. Real-time PCR method was used to detect the levels of C4 transcripts (A). L-O2 cells was transiently transfected with the full-length pC4 -1,007/+75 (p-C4) plasmid and then treated with WGP or vehicle control for 72 h. Luciferase assays were performed to determine the C4 promoter activity (B). Data are presented as mean of triplicates ±SEM from one representative experiment. * indicates p < 0.05, ** indicates p < 0.005, and *** indicates p < 0.001 compared to vehicle control. ### indicates p < 0.001 compared to the basic vector control.

FIGURE 6

WGP enhance C4 transcription via the E-box1 and Sp1 elements. (A) The critical cis-regulatory elements (NF-1, E-box1∼3, Sp1) on the plus (+) and minus (-) DNA strand are shown. Numbering is relative to the C4 translation start site. A series of 5’-deletion constructs were generated by PCR amplification and subcloning into the pGL4.19 basic vector. (B) The C4 promoter constructs were transiently transfected into L-O2 cells. Luciferase assay was used to measure the activity of C4 promoter. * indicates p < 0.05, ** indicates p < 0.005, and *** indicates p < 0.001 compared to no drug treatment control. ### indicates p < 0.001 compared to basic vector control. (C) Fold changes of WGP treatment compared to vehicle control are graphed. * indicates p < 0.05 and ** indicates p < 0.005. Data are presented as mean of triplicates ±SEM from one representative experiment.