TW201306854A - Anti-respiratory syncytial virus of immunomodulatory protein isolated form Flammulina velutipes and Ganoderma tsugae - Google Patents

Abstract

The most common agent of lower respiratory infections in early childhood is respiratory syncytial virus (RSV). Because there is no clear mechanism of the infection, the effective methods of therapy are not easy to develop. FIP-fve and FIP-gts are a family of immunomodulatory proteins isolated from Flammulina velutipes and Ganoderma tsugae, respectively. The molecular weight of FIPs is about 13 kDa. These family proteins stimulated the production of IFN- γ and reduced the allergic inflammation in human PBMCs. In this claim, we detect the quantity of RSV on HEp-2 cells by RT-PCR, plaque assay, Immunofluorescence assay and ELISA after FIP-fve treatment and find that FIP-fve may suppress the infection of RSV. In the claim, we detected the proteins of phospho-NF- κ B and phosphor-I κ B α with Western blotting and observed the translocation of NF- κ B in A549 cells. These results demonstrate that FIP-fve reduce the phosphorylation of NF- κ B and I κ B α and the translocation of NF- κ B. Furthermore, the inflammatory cytokines are decreased in HEp-2 cells with FIP-fve treatment. In vivo, oral FIP-fve and FIP-gts could alleviate on RSV-induced airway inflammation. They are developed to therapeutic agents for viral airway disease.

Description

Development of Flammulina velutipes immunomodulatory protein and Ganoderma lucidum immunomodulatory protein for inflammation caused by respiratory syncytial virus and reduction of virus dose

Biotechnology, agrochemistry

1. Introduction of Respiratory Syncytial Virus (RSV)

Respiratory Syncytial Virus (RSV) is the leading cause of acute viral respiratory diseases such as viral bronchitis and viral pneumonia in infants and young children [1]. It is an RNA virus, and the mumps virus. The German measles virus belongs to the same Paramyxovirus. It can produce a special cell fusion phenomenon (syncytial cytopathology) in a variety of different tissues. It can cause necrosis of the tracheal epithelial cells after invading the respiratory tract. Mucus secretion, inflammatory cell infiltration, submucosal edema [2]. Respiratory fusion virus particularly likes to infect the epithelial cells of the respiratory tract with cilia, which can cause obstruction and inflammation of the bronchioles and infiltration of submucosal cells in the respiratory tract, prolonged expiratory time, inelectasis and pneumonia. In the elderly and immunocompromised patients, respiratory syncytial virus can also cause serious respiratory diseases. From the perspective of public health, health organizations and government health units around the world have made the production of respiratory syncytial virus vaccines one of the most important tasks in the prevention and treatment of viral infections.

The respiratory syncytial virus is Paramyxoviridae, a Pneumovirus having an envelope, the genome of which is a single-stranded (-) RNA consisting of 15,222 nucleic acids and comprising 10 segments of mRNA. These mRNAs can be translated into 11 proteins that have been identified, including four nucleocapsid proteins, named nucleocapsid N protein, phosphoprotein P, large polymerase subunit transcription elongation factor M2-1; The coat protein of the membrane is fusion F protein, attachment G protein and small hydrophobic SH protein [3]; two kinds of non-structural proteins, NS1, NS2; one matrix protein M protein and M2- currently speculated as negative regulator 2 and other proteins. Currently, NS1, NS2 and SH proteins are not well defined. Based on the results of experiments conducted by scientists in the past for the development of respiratory syncytial virus vaccines, we can conclude that the immune response elicited by the inactivated vaccine is a series of reactions of Th 2 T-helper cells that cause the lower respiratory tract (referred to as the bronchi, lungs). The condition of the other parts is more serious, because the cytokines IL-4, IL-10 produced by Th 2 T-helper cell cause more serious cell infiltration, aggravate and accelerate the disease. In the epidemiological survey of northern Taiwan, 2001/01-2005/12, the spring and autumn of every two years is the peak of the disease (Lee et al., 2007). Currently, there are no specific drugs for supportive therapy. And treatment with glucocorticoids does not improve their symptoms (Patel et al., 2008). In the implementation of this study, we found that FIP-fve may induce Th 1 T-helper cell response that effectively inhibits viral infection and relieves respiratory diseases. It is an effective method to develop clinical drugs that meet our requirements.

Second, the function of fungal immune regulatory proteins

In some edible mushrooms, such as: Ganoderma lucidum, straw mushroom and Flammulina velutipes, they have similar amino acid sequences and immunomodulatory functions. We named this type of protein as a fungal immune regulatory protein. (Fungal immunomodulatory proteins, FIPs) (Hsu et al., 1997; Ko et al., 1995; Lin et al., 1997). In particular, Ganoderma lucidum has become a leader in traditional Chinese medicine and has a great effect on maintaining human health. Previous studies have found that it has anti-allergic inflammation (Lee et al., 2001), protects liver function (Shieh et al., 2001), anti-tumor and enhances immune function, but is mostly limited to crude extracts (Cuella et al., 1996). Research, or small molecule compound research (Lai et al., 2001; Zhang et al., 2002). Until 1989, Meiji Pharmaceutical of Japan purified an immunoregulatory protein, LZ8 (Kino et al., 1989), from Ganoderma lucidum. The results showed that LZ-8 can significantly inhibit systemic allergic reactions, treat hepatitis and prevent diabetes. Recently, Ganoderma lucidum immunoregulin has been found to inhibit the terminal enzyme activity of lung cancer cells (Liao et al., 2006; Liao et al., 2007) and cause lung cancer cell senescence (Liao et al., 2008) to develop lung cancer and assist probiotics. Technology Development Co., Ltd. applied for a patent. LZ8 is mitogenic and inhibits anaphylaxis caused by bovine serum albumin BSA in CFW mice. The host of this program has found a similar degree of similarity to the protein structure of the heavy chain region of LZ8 and immunoglobulin from another edible mushroom, Flammulina velutipes (FIP-fve), and the secondary structure is mostly b-Sheet. When the ability to proliferate human lymphocytes was measured, when the concentration of FIP-fve was 100 μg/ml (Ko et al., 1995), the proliferative ability was the strongest. Injection of FIP-fve protein in mice at a dose of 8 mg/kg body weight inhibited the systemic anaphylaxis response caused by BSA. Pre-intraperitoneal injection of FIP-fve protein at a dose of 5 mg/kg body weight in the mouse toe swelling test reduced the inflammatory response caused by compound 48/80. From the above experimental results, it was found that the protein has immunomodulatory activity, and further studies on the regulation of immune regulation are to promote the transcription of IFN-γ and TNF-α genes.

Among them, the true scorpion immunomodulatory function protein is more transparent than Ganoderma lucidum. This immunoregulatory protein promotes the expression of ICAM-1 (intercellular adhesion molecule-1) (Haak-Frendscho et al., 1993) and significantly increases IFN. -γ, so Ganoderma lucidum increases CD2 expression and causes the formation of rosette, so Ganoderma lucidum can express its medicinal properties by regulating the adhesion molecules on the immunocompetent cell. Among the strains of Ganoderma lucidum, several kinds of Ganoderma lucidum, such as G. applanatum., G. formosanum., G. fornicatum., G. lucidium, G. tropicum and G. tsugae, were identified by Dr. Xu Xuxiang. The immunomodulatory activity varies greatly between each species. Currently, G. lucidium and G. tsugae and the FIP-fve discovered by the researchers have been confirmed to have immunomodulatory functions, and the research team has also developed new immunity this year. Regulate functional protein ability.

In mosses, some of their fruiting bodies or mycelium are in mosses, and their fruiting bodies or mycelium have some lectins that promote lymphocyte proliferation; Some will inhibit the proliferation of lymphocytes. Including Agaricus bisporus (Koyama et al., 2002), with a molecular weight of 64 kDa, consisting of four subunits, inhibits proliferation of cancer cells U937 and even causes apoptosis (Apoptosis). These glycoproteins all have the ability to aggregate cells. The glycosidic protein in steroids has many different carbohydrates bound to it. The saccharide on Hericium erinaceum (Kawagishi et al., 1994) belongs to sialic acid binding, Laetiporus sulfurens (Yoshikawa et al., 2001) is a combination of N-acetyllactosamine, which may promote the binding of cell surface receptors, while the glycoprotein has different binding ability to different kinds of antibodies or cells for immunomodulatory functions, such as: Agaricus bisporus Ig A binding (Irazoqui et al., 1997); L-selectin promotes neutrophil agglutination; C. albicans purified manoprotein also reduces IL-8 binding to IL-8 receptor and slows inflammatory response caused by IL-8 (Kemeny et al., 1998). Therefore, this study is mainly to explore whether such agglutinin can reduce the immune function of immune regulation and reduce the damage of RSV.

3. Flammulina velutipes immunoregulatory protein and Ganoderma lucidum immunomodulatory protein

The protein obtained by purifying the Flammulina velutipes fruit body via the cation exchange resin CM-52: FIP-fve (Fungal immunomodulatory protein) is a protein having an immunoregulatory function. It is known from previous experiments that FIP-fve can induce the production of IFN-γ by lymphocytes (HPBMCs) of normal human peripheral blood and spleen cells of Balb/c mice, and has the ability to proliferate cells. The IFN-γ induced by FIP-fve is a cytokine induced by Th1 cells and can inhibit the cytokines produced by Th2 cells. The recombinant Ganoderma lucidum immunomodulatory protein was further purified by placing the Ganoderma lucidum immunoregulatory gene into Baker's yeast to obtain a 13 kDa protein. Therefore, it has an allergic reaction on the immune system which reduces the regulation of IgE, mast cell, etc. by Th2 cytokine, including chronic asthma, respiratory inflammation and the like. Therefore, in this study, FIP-fve (100μg/ml) stimulated HPBMCs to phosphorylate p38 and ERK (extracellular signal-regulated protein kinase), but did not activate JNK (thec-Jun NH2-terminal kinase), and pre-treated Inhibitor of p38 MAP kinase: SB203580 (10 μM) inhibits phosphorylation of p38. It was also demonstrated that treatment of HPBMCs with FIP-fve (100 μg/ml) for 48 hours resulted in significant production of IFN-γ, and induction was also inhibited by SB203580, LY294002 (inhibitor of PI3-kinase) and U0126 (inhibitor of MEK1/2). . In addition to the ability of FIP-fve protein purified from the velutipes fruit body to induce IFN-γ, the recombinant FIP-fve fusion protein (recombinant FIP-fve) also has nearly half of the activity of FIP-fve. This experiment demonstrates that the mechanism by which FIP-fve regulates IFN-γ is through the p38 MAP kinase pathway and the activation pathway of ERK, rather than via the activated JNK pathway. The high amount of IFN-γ induced by FIP-fve can inhibit the production of cytokines produced by TH2 cells, and it is expected to be applied to the prevention and treatment of genetic immunity in the future.

Development of Flammulina velutipes immunomodulatory protein for the treatment of respiratory syncytial virus activity

Plaque assay

2. Immunofluorescence staining (Immunofluorescence)

3. Enzyme immunosorbent assay (ELISA)

4. Polymerase chain reaction (RT-PCR)

5. Experimental animals: 7-week-old BALB/c mother

It is provided by the National Animal Center of the Foundation and is raised at the Experimental Animal Center of Zhongshan Medical University.

6. BUXCO SYSTEM-AHR

Figure 1 and the left panel show the results of purification on a 12% SDS PAGE gel. The molecular weight of Flammulina velutipes is 13 kDa. M represents the marker, C represents the crude extract, and fve represents the purified Flammulina velutipes immunomodulatory protein FIP-fve.

Figure 2. The effect of Flammulina velutipes and Ganoderma lucidum immunomodulatory proteins on cell plaques caused by RSV was observed by plaque counting test. In a 12-well culture dish, 3×10 ⁵ HEp-2 cells were treated with different concentrations of protein for 2 hours, and then RSV was infected for 1 hour and covered with 0.75% methylcellulose for 6 days. The situation of plaque formation was observed by crystal violet staining.

Figure 3. The effect of FIP-fve on RSV infectivity was observed by immunofluorescence staining. Two hours after pretreatment of 200 μg/ml Flammulina velutipes in human lung cancer epithelial A549, different doses of RSV were infected for 48 hours, and RSV was detected by specific fluorescent antibody. The blue fluorescent part was nucleus, green fluorescent For RSV.

Figure 4. The effect of FIP-fve on RSV infectivity was assessed by enzyme immunosorbent assay. After pretreatment of 0, 50, 100, 200 μg/ml Flammulina velutipes protein in human lung cancer epithelial A549 for 2 hours, different doses of RSV were infected. After 72 hours, the cells were fixed with acetone and bound with specific RSV antibody. The absorbance is detected at a wavelength of 450 nm.

Figure 5. Detection of RSV and IL-4 mRNA expression by RT-PCR. After pretreatment with 0,50,100 μg/ml Flammulina velutipes protein in human laryngeal carcinoma cell line HEp-2 for 2 hours, RSV was infected (moi=0.1). After 48 hours, mRNA was purified and detected by RT-PCR. Its RSV and IL-4 gene expression levels.

Figure 6. Detection of IL-6 secretion by ELISA. After pretreatment of 0,100,400 μg/ml Flammulina velutipes protein in human laryngeal carcinoma cell line HEp-2 for 2 hours, RSV (moi=3) was infected again. After 24 hours, the culture medium was collected and detected by ELISA. -6 concentration.

Figure 7. The effect of FIP-fve on RSV-induced phosphorylation of NF-κB and IκBα by Western blotting. RSV was infected in A549 lung cancer epithelial cells, and 200 μg/ml Flammulina velutipes protein was administered. The total protein in the cells was isolated after 4 hours and 8 hours, respectively, and phosphorylated NF-κB (p-NF- was observed by Western blotting method. κB) and protein expression of phosphorylated IκBα (p-IκBα).

Figure 8. Immunofluorescence staining was used to observe the effect of FIP-fve on the entry of NF-κB into the nucleus after RSV infection. Two hours after pretreatment of 200 μg/ml Flammulina velutipes in human lung cancer epithelial A549, different doses of RSV were infected for 24 hours, and RSV was detected by specific fluorescent antibody. The blue fluorescent part was nucleus, red fluorescent For NF-κB.

Figure 9. After stimulation with RSV, the IKK complex in the cell phosphorylates IκB, while phosphorylated IκB degrades and releases NF-κB. On the other hand, NF-κB is also phosphorylated by IKK complex. And into the nucleus, NF-κB enters the nucleus and binds to the transcriptional sites of many inflammatory substances (eg, IL-4), and the gene expression is activated, causing the infected cells to produce an inflammatory response. The results of this study suggest that FIP-fve may have the potential to reduce RSV infection, thereby reducing the production of inflammatory substances.

Figure 10. Observing the effect of Flammulina velutipes and Ganoderma lucidum immunoregulatory protein on respiratory tract overreaction in RSV-infected mice. Each mouse was orally administered with 200 ug of Flammulina velutipes protein or 50 μg of Ganoderma lucidum protein once every 12 hours. After 48 hours, 10 ⁸ PFU of RSV virus was administered by nasal titration, and oral protein was continuously administered. On the 6th day after sensitization, Mice were exposed to misty methacholine at concentrations of 0 (saline), 5, 10, 20 and 40 mg/ml, respectively, and subjected to a mouse airway hyperresponsiveness assay (AHR).

Figure 11. The effect of Flammulina velutipes and Ganoderma lucidum immunomodulatory proteins on the IL-6 content in the lungs of RSV-infected mice. Each mouse was orally administered with 200 μg of Flammulina velutipes protein or 50 μg of Ganoderma lucidum protein every 12 hours. After 48 hours, 10 ⁸ PFU of RSV virus was administered by nasal titration, and oral protein was continuously administered. After sensitization, it was collected on the 6th day. Mouse lung lavage fluid and IL-6 concentration was detected by ELISA.

AHR. . . Respiratory hyperresponsiveness

Methacholine (mg/ml). . . Acetylcholine

Penh. . . Pulmonary function index

Control. . . Control amount

RSV. . . Respiratory fusion virus

FIP-fve/RSV. . . Immunomodulatory protein

FIP-gts/RSV. . . Ganoderma lucidum protein

Claims (3)

Oral true steroid immunoregulatory protein, including: Flammulina velutipes immunoregulatory protein or Ganoderma lucidum immunomodulatory protein can inhibit respiratory tract hyperactivity caused by RSV. Flammulina velutipes immunoregulatory protein inhibits RSV-induced phosphorylation of NF-κB and enters the nucleus. Oral administration of Flammulina velutipes immunoregulatory protein can inhibit the inflammatory response-related hormones induced by RSV and reduce the amount of virus expression.