A Study On Immunomodulatory Mechanism Of Polysaccharopeptide Mediated By TLR4Signaling Pathway

Objective: To study the effects of polysaccharopeptide (PSP) onTLR4signaling pathway in mice peritoneal macrophages and the spleens ofEAC tumor-bearing mice, and explore the immunomodulatory mechanismof PSP both in vitro and in vivo.Methods:1. After isolation of peritoneal macrophages from C57BL/10J mice(wild-type mice with functional TLR4) or C57BL/10ScCr mice(TLR4-defient mice lacking functional TLR4), cells were divided into threegroups: control group, PSP group (25μg/ml) and positive control LPS group(100ng/ml). The concentrations of TNF-α and IL-6in supernatants weredetected by ELISA. Western blot was used to detect the related proteinexpression of TLR4pathway in peritoneal macrophages from each group.Meanwhile, Q-PCR and Western blot were used to detect the related geneand protein expression of TLR4pathway regulated by PSP.2. Ehrlich’sascites carcinoma (EAC) C57BL/10J mice (wild-type micewith functional TLR4) or C57BL/10ScCr mice (TLR4-defient mice lackingfunctional TLR4) were used to establish the animal model for solid tumor. Mice were randomly divided into four groups: NS group, Adriamycin(ADM) group, PSP group and LPS group. After25days of treatment, micewere sacrificed by cervical dislocation, and the whole body, tumor, spleenand thymus were weighed immediately. The inhibition rates of growth ofEAC solid tumor and the immune organ index were calculated respectively.Quantitative real-time PCR (Q-PCR) and Western blot were used to detectthe related gene and protein expression of TLR4pathway in spleens fromeach group.Results:1. In vitro, LPS and PSP induced TNF-α and IL-6secretion byperitoneal macrophages from wild-type control B10(TLR4+/+) mice (P <0.05) but did not induce TNF-α and IL-6secretion in ScCr(TLR4-/-) micelacking functional TLR4(P>0.05). The protein levels of TLR4and TRAF6were upregulated in PSP and LPS group compared with control group inperitoneal macrophages of B10(TLR4+/+) mice (all P <0.05). Thephosphorylation levels of transcription factors NF-κB (p65subunit) andAP-1were likewise significantly higher in PSP and LPS group comparedwith control group in peritoneal macrophages of B10(TLR4+/+) mice (all P <0.05). No significant differences in those protein expressions were foundamong groups in ScCr (TLR4-/-) mice (all P>0.05).2. In vivo, the mean weights of tumors in B10(TLR4+/+) tumor-bearingmice that received PSP and ADM treatment were significantly decreased than NS group (all P <0.05). The inhibition rates of ADM group and PSPgroup were40.68%and25.20%. The thymus index in the ADM groupdecreased significantly, as compared with that in the NS group (P <0.05).Both the thymus index and spleen index in the PSP group increasedsignificantly relative to that in the ADM group (all P <0.05). In ScCr(TLR4-/-) tumor-bearing mice, the mean weights of tumors in the ADMgroup was decreased significantly compared with that in the NS group (P <0.05)，the inhibition rate of ADM group was38.21%. However, there wereno significant change trends between PSP group and NS group (p>0.05)and there were no significant differences of the thymus index and spleenindex among groups in ScCr (TLR4-/-) mice (P>0.05). The mRNA andprotein levels of TLR4, TRAF6, NF-κB and AP-1in PSP and LPS group(positive control) were significantly increased relative to those of the NSgroup in the spleen of B10(TLR4+/+) tumor-bearing mice (all P <0.05). Asexpected, there were no significant differences in the expressions of TLR4,TRAF6, NF-κB and AP-1among groups in ScCr (TLR4-/-) mice (all P>0.05).Conclusions: In vitro and in vivo data suggest that PSP may exert itsimmunomodulatory potency via TLR4signaling pathway.