Polysaccharides from Pinus massoniana pollen improve intestinal mucosal immunity in chickens.

Intestinal mucosa is the largest immune organ in animals, and its immune function is directly related to the resistance against various diseases. Taishan Pinus massoniana pollen polysaccharides (TPPPS) have been recognized as an effective vaccine adjuvant and potential immune enhancer against viral infections. However, little is known about their direct immune-enhancing activity on intestinal mucosa. In this study, we extracted the polysaccharides from Taishan masson pine pollen to investigate its promotive effect on intestinal mucosal immunity. A total of 120 1-day-old chickens were divided into 4 groups and inoculated with PBS or 3 different doses of TPPPS (10 mg/mL, 20 mg/mL, and 40 mg/mL), respectively. Feces, intestinal specimens, and serum samples were collected from the chickens at 7, 14, and 21 d after inoculation. The antibodies in serum, mucosal secretion of IgA, structure of intestinal villi, and expressions of cytokine genes and mucosal immune-related genes in the chickens were all significantly improved by TPPPS treatments. At 21 d after inoculation following the challenge of Newcastle disease virus, the chickens inoculated with 20 and 40 mg/mL TPPPS exhibited decreased weight loss and reduced intestinal pathologic damage and viral loads in the intestine. In summary, our results demonstrate that TPPPS can enhance mucosal immunity and promote intestinal villi development. This study has established the foundation for the development of novel immune-enhancing agent with immune-regulatory effects on intestinal mucosa.

Effects of Pinus massoniana pollen polysaccharides on intestinal microenvironment and colitis in mice.

The stability of the intestinal microenvironment is the basis for maintaining the normal physiological activities of the intestine. On the contrary, disordered dynamic processes lead to chronic inflammation and disease pathology. Pinus massoniana pollen polysaccharide (PPPS), isolated from Taishan Pinus massoniana pollen, has been reported with extensive biological activities, including immune regulation. However, the role of PPPS in the intestinal microenvironment and intestinal diseases is still unknown. In this work, we initiated our investigation by using 16S rRNA high-throughput sequencing technology to assess the effect of PPPS on gut microbiota in mice. The result showed that PPPS regulated the composition of gut microbiota in mice and increased the proportion of probiotics. Subsequently, we established immunosuppressive mice using cyclophosphamide (CTX) and found that PPPS regulated the immunosuppressive state of lymphocytes in Peyer's patches (PPs). Moreover, PPPS also regulated systemic immunity by acting on intestinal PPs. PPPS alleviated lipopolysaccharide (LPS) -induced Caco2 cell damage, indicating that PPPS has the ability to reduce the damage and effectively improve the barrier dysfunction in Caco2 cells. In addition, PPPS alleviated colonic injury and relieved colitis symptoms in dextran sodium sulfate (DSS)-induced colitis mice. Overall, our findings indicate that PPPS shows a practical regulatory effect in the intestinal microenvironment, which provides an essential theoretical basis for us to develop the potential application value of PPPS further.

Screening active fractions from Pinus massoniana pollen for inhibiting ALV-J replication and their structure activity relationship investigation.

The objective was to identify the active fractions of polysaccharide against replication of ALV-J and elucidate their structure activity relationship. The optimal extraction conditions were extracting temperature 90℃, pH 9 and the ratio of liquid to solid 30:1. Under these conditions, extraction yield of total polysaccharide was 6.5 % ± 0.19 %. Total polysaccharide was then purified by DEAE-52 cellulose and Sephadex G-200 gel. Three fractions, PPP-1, PPP-2, and PPP-3, were identified with molecular weight of 463.70, 99.41, and 26.97 kDa, respectively. Three polysaccharide fractions were all composed of 10 monosaccharides in different proportions. Compared with PPP-1, which was mainly composed of glucose, PPP-2 and PPP-3 contained a higher proportion of galactose, glucuronic acid and galacturonic acid. The Congo red assay indicated that the PPP-2 may have a triple helical structure, while PPP-1 and PPP-3 were absent. In vitro assay showed that there was no significant cytotoxicity among the polysaccharide fractions under the concentration of 800 µg mL-1 (P > 0.05). The antiviral test showed that PPP-2 had the strongest activity, indicating PPP-2 was the major antiviral component. The structure-activity relationship showed that the antiviral activities of polysaccharide fractions were affected by their monosaccharide composition, molecular weight, and triple helical structure, which was a result of a combination of multiple molecular structural factors. These results showed that the PPP-2 could be exploited as a valued product for replacing synthetic antiviral drugs, and provided support for future applications of polysaccharide from Pinus massoniana pollen as a useful source for antiviral agent.

Taishan Pinus massoniana pollen polysaccharide inhibits H9N2 subtype influenza virus infection both in vitro and in vivo.

The H9N2 subtype avian influenza virus (AIV) is one of the most prevalent AIV subtypes that can be found throughout most countries. Currently, due to the neglect of low pathogenic avian influenza virus (LPAIV) and monotonous control technique, an expanding H9N2 virus epizootic have been arisen and causes great economic losses in the poultry industry. Therefore, novel anti-influenza drugs are necessary for the prevention and control of H9N2 AIV. Our previous studies have found that Taishan Pinus massoniana pollen polysaccharides (TPPPS) have antiviral effects, but whether they can inhibit the H9N2 AIV remains unclear. Here, we further investigated the effects of TPPPS on the H9N2 virus and its underlying mechanisms of action. We found that TPPPS significantly inhibited the replication of the H9N2 virus in a dose-dependent manner, especially during the period of virus adsorption in vitro. Transmission electron microscopy demonstrated that TPPPS reduce infection by interfering with virus entry into host cells rather than by interacting with the H9N2 virus particles. A fluorescence quantitative PCR (qPCR) assay and an animal experiment were performed to evaluate the anti-viral effect of TPPPS in vivo. As expected, the lungs of chickens treated with TPPPS had fewer lesions and lower virus contents compared with the PBS group. In addition, pre-treatment with TPPPS clearly enhanced host disease resistance and delayed infection by the H9N2 virus. Taken together, our results reveal that TPPPS suppress H9N2 virus replication both in vitro and in vivo and therefore shows promising as an anti-AIV agent.

Taishan Pinus Massoniana pollen polysaccharide inhibits the replication of acute tumorigenic ALV-J and its associated tumor growth.

Avian leukosis virus subgroup J (ALV-J) has resulted in considerable economic losses in the poultry industry. In recent years, fibrosarcoma induced by ALV-J, which contains the v-fps oncogene, has gained momentum, and this has brought about new challenges to the poultry industry. To study the inhibitory effects of Taishan Pinus Massoniana pollen polysaccharide (TPPPS) on acute ALV-J infection and tumor development, antiviral and antitumor models of the Fu-J (SDAU1005) strain of ALV-J were established in vitro and in vivo. The results of in vitro experiments showed that TPPPS significantly inhibited viral replication in a dose-dependent manner during adsorption and pretreatment stages. The results of in vivo experiments have shown that TPPPS significantly reduced the viral load in the plasma and tumor tissues, as well as inhibited tumor growth. We further examined the difference in transcriptome expression by using RNA-Seq technology. A total of 560 differentially expressed genes were identified that included 329 up-regulated genes and 231 down-regulated genes. The up-regulated genes were mainly immune-related genes, whereas the down-regulated genes were mainly tumor-regulated genes. Gene Ontology (GO) term enrichment included immune system processes, positive regulation of immune system processes, regulation of immune system processes, leukocyte activation, cell activation, and protein binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the main immune and tumor-related pathways included T-cell receptor signaling pathway, cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, NF-κB signaling pathway, and Ras signaling pathway. In summary, our results preliminarily point to the antiviral and antitumor mechanism of TPPPS in vivo and in vitro.