Lilly Ganju

Immunomodulatory effects of agents of plant origin

The immunomodulatory properties of amla (Emblica officinalis) and shankhpushpi (Evolvulus alsinoides) were evaluated in adjuvant induced arthritic (AIA) rat model. Injecting Complete Freunds Adjuvant (CFA) in right hind paw of the animals induced inflammation. The crude extracts of both the herbs were administered intraperitonially following a repeated treatment profile. The anti-inflammatory response of both the extracts was determined by lymphocyte proliferation activity and histopathological severity of synovial hyperplasia. Both the extracts showed a marked reduction in inflammation and edema. At cellular level immunosuppression occurred during the early phase of the disease. There was mild synovial hyperplasia and infiltration of few mononuclear cells in amla or shankhpushpi treated animals. The induction of nitric oxide synthase (NOS) was significantly decreased in treated animals as compared to controls. These observations suggest that both the herbal extracts caused immunosuppression in AIA rats, indicating that they may provide an alternative approach to the treatment of arthritis.

EFFECT OF HIPPOPHAE RHAMNOIDES LEAF EXTRACT AGAINST DENGUE VIRUS INFECTION IN HUMAN BLOOD-DERIVED MACROPHAGES

Dengue virus occurs as four distinct serotypes, called Dengue 1, 2, 3, and 4. Symptomatic dengue virus infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The anti-Dengue treatment is severely hampered as no specific therapeutic agents are available. Even present treatment strategies for Dengue are more supportive than curative. In the present study anti-dengue activity of Hippophae rhamnoides (Seabuckthorn, SBT) leaf extract was evaluated in Dengue virus type-2 infected blood-derived human macrophages as macrophages are the primary target of Dengue virus infection. Infected cells were treated with SBT leaf extract and compared with commercially available anti-viral drug, Ribavirin. The extract was able to maintain the cell viability of Dengue-infected cells at par with Ribavirin along with the decrease and increase in TNF-alpha and IFN-gamma respectively. Anti-dengue activity of SBT extract was further determined by the traditional plaque assay. These observations suggest that the SBT leaf extract has a significant anti-dengue activity and has the potential for the treatment of Dengue.

Aqueous Extract of Rhodiola imbricata Rhizome Stimulates Proinflammatory Mediators via Phosphorylated IκB and Transcription Factor Nuclear Factor-κB

Modulation of immune response to alleviate diseases has long since been of interest. Plant extracts have been widely investigated for their possible immunomodulatory properties. We have evaluated the immunomodulatory activity of aqueous extract of Rhodiola rhizome in human peripheral blood mononuclear cells (PBMCs) and mouse macrophage cell line RAW 264.7. The Rhodiola extract was found to stimulate production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in human PBMCs as well as RAW 264.7 cell line. It also increased production of nitric oxide synergistically in combination with lipopolysaccharide (LPS) in RAW 264.7. Rhodiola at 250 μg/ml increased the p-IκB expression in human PBMCs. Aqueous extract of Rhodiola (250 μg/ml) also activated the nuclear translocation of NF-κB in human PBMCs, which is comparable to the positive stimulant LPS. Thus, our present study suggests that Rhodiola most likely activates proinflammatory mediators via phosphorylated inhibitory kB and transcription factor NF-kB. Our study demonstrates immunostimulatory potential of aqueous extract of Rhodiola rhizome, that can be used for upregulation of immune response in patients with inadequate functioning of the immune system.

Effect of Seabuckthorn (Hippophae rhamnoides) flavone on immune system: an in-vitro approach.

There are several reports, which suggest that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavones, of Seabuckthorn (SBT) (Hippophae rhamnoides L.) fruit berry can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines. We have evaluated the immunomodulatory activity of ethanolic solution of SBT flavone (FLV) in human peripheral blood mononuclear cells (PBMCs). The SBT flavone was found to stimulate production of interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) in PBMCs. However, increased expressions of p‐IκB, NF‐κB, and p‐p38 were found in flavone‐treated human PBMCs with significantly suppressed expression of CD25 (IL‐2R). There was no alteration found in the nitric oxide (NO) production in mouse macrophage cell line RAW 264.7. These observations suggest that stimulation of IL‐6 and TNF‐alpha secretion may contribute to the putative beneficial effects of dietary flavone against microbial infection. Copyright

Aqueous extract of Rhodiola imbricata rhizome inhibits proliferation of an erythroleukemic cell line K-562 by inducing apoptosis and cell cycle arrest at G2/M phase.

Rhodiola imbricata is a medicinal plant having immunostimulating properties. The anti-proliferative effects of Rhodiola aqueous extract (RAE), were studied in human erythroleukemic cell line K-562 using MTT cell proliferation assay. The proliferation of K-562 was significantly decreased after 72h incubation with RAE at 100 and 200microg/ml. However, almost no suppressive effects could be detected in normal human peripheral blood lymphocytes or mouse macrophage cell line RAW-264.7. RAE was also found to induce intracellular reactive oxygen species (ROS) in K-562 cells at 200microg/ml when incubated overnight. The increased ROS generation may cause apoptosis, which was observed in AnnexinV-FITC and propidium iodide (PI) staining of cells treated with RAE for 72h in K-562 cells. Moreover, RAE arrested cell cycle progression in G2/M phase in early and late period of exposure. The anti-cancer activity of RAE was also confirmed by increased NK cell cytotoxicity. These observations suggest that aqueous extract of R. imbricata rhizome has very potent anti-cancer activities, which might be useful in leukemia cancer treatment.

Altered expression of platelet proteins and calpain activity mediate hypoxia induced prothrombotic phenotype

Oxygen-compromised environments, such as high altitude, air travel, and sports, and pathological conditions, such as solid tumors, have been suggested to be prothrombotic. Despite the indispensable role of platelets in thrombus formation, the studies linking hypoxia, platelet reactivity, and thrombus formation are limited. In the present study, platelet proteome/reactivity was analyzed to elucidate the acute hypoxia-induced prothrombotic phenotype. Rats exposed to acute simulated hypoxia (282 torr/8% oxygen) demonstrated a decreased bleeding propensity and increased platelet reactivity. Proteomic analysis of hypoxic platelets revealed 27 differentially expressed proteins, including those involved in coagulation. Among these proteins, calpain small subunit 1, a 28-kDa regulatory component for calpain function, was significantly upregulated under hypoxic conditions. Moreover, intraplatelet Ca(2+) level and platelet calpain activity were also found to be in accordance with calpain small subunit 1 expression. The inhibition of calpain activity demonstrated reversal of hypoxia-induced platelet hyperreactivity. The prothrombotic role for calpain was further confirmed by an in vivo model of hypoxia-induced thrombosis. Interestingly, patients who developed thrombosis while at extreme altitude had elevated plasma calpain activities and increased soluble P-selectin level. In summary, this study suggests that augmented calpain activity is associated with increased incidence of thrombosis under hypoxic environments.

RNA interference mediated silencing of Hsp60 gene in human monocytic myeloma cell line U937 revealed decreased dengue virus multiplication

Heat shock proteins (Hsps) or stress proteins are highly conserved molecules and expressed in all cell types under stressful conditions like heat, cold, hypoxia and infections. The objective of the present study was to determine the effect of dengue virus infection on relative expression of stress proteins and their role in the progression of the infection. As macrophages are the primary host for dengue, human promonocytic myeloblastoma U937 cells were infected with dengue virus type 2 New Guinea C strain for the evaluation of Hsps expression. A significant expression of Hsp60 was observed in virally infected U937 cells as compared to controls. In order to determine the correlation between Hsp60 expression and viral multiplication in infected cells, expression of Hsp60 was down regulated by RNA interference. Viral multiplication was determined by quantification of viral RNA copy number using Real Time PCR and plaque formation assay in cellular supernatants of Hsp60 silenced cells. Intracellular quantification of viral load was also determined by flow cytometry. It was observed that down regulation of Hsp60 in virally infected cells resulted into decrease in viral RNA copy number, plaque forming units and intracellular viral load. At the same time down regulation also resulted in increased IFN-alpha level. These observations suggest that, elevated levels of Hsp60 expression in virally infected cells may help in viral multiplication and could be possible therapeutic targets for the management of dengue virus infection.

Aqueous extract of Rhodiola imbricata rhizome stimulates Toll-like receptor 4, granzyme-B and Th1 cytokines in vitro

Rhodiola imbricata is a medicinal plant, native to mountainous regions of Asia, parts of Europe, and the Arctic. Traditionally it is recommended to help combat fatigue and restore energy. It exhibits anti-stress, anti-cancer, and immunostimulatory activities. However, the effect of Rhodiola on immunological responses largely remains unknown. In this study, we have investigated the effect of aqueous extract of R. imbricata rhizome (RAE), on Toll-like receptor-4 (TLR-4) and intracellular granzyme-B expression in mouse splenocytes. Furthermore, TH1/TH2 cytokine profile was analyzed in RAE-treated human peripheral blood mononuclear cells (PBMCs) using multiplex flowcytomix kit. Our findings suggest that RAE induces TLR-4 expression and intracellular granzyme-B in treated splenocytes while RAE stimulated IL-1beta, IL-6, and TNF-alpha in human PBMCs. The present study suggests that RAE stimulates the innate immune pathway and has potent immunostimulatory activity, which can be used in modulating the immune system of immunocompromised individuals.

Dengue virus infection induces upregulation of hn RNP-H and PDIA3 for its multiplication in the host cell

The pathogenic mechanism of Dengue virus (DENV) infection is related to the host responses within target cells and therefore, we assessed intracellular changes in host cell proteins following DENV infection. This study provides evidence that heterogeneous nuclear ribonucleoprotein H (hnRNP-H) and protein disulfide isomerase A3 (PDIA3) helps in DENV multiplication by suppressing TNF-α production in human monocytic THP1 cells. Proteomic analysis of infected cells, identified upregulation of the host cell proteins PDIA3 and hnRNP-H in comparison to mock infected cells. The functional role of hnRNP-H and PDIA3 in DENV infection was identified by down regulating hnRNP-H and PDIA3 genes with their specific siRNA duplexes which lead to decreased intracellular viral load. It also resulted in increased TNF-α level which mediates antiviral effect. This is the first study, which reports the role of PDIA3 and hnRNP-H in TNF-α production in DENV infected cells. Collectively, these results suggest that increased level of hnRNP-H and PDIA3 expression in DENV infected THP1 cells assist in the viral replication by suppressing the TNF-α production.

Dengue Virus Infection Activates Cellular Chaperone Hsp70 in THP-1 Cells: Downregulation of Hsp70 by siRNA Revealed Decreased Viral Replication

The pathogenic mechanism of dengue virus infection is related to the host responses within target cells, and therefore we assessed intracellular changes in stress proteins following dengue virus infection. This study provides evidence that Hsp70 helps in viral multiplication by suppressing the type 1 interferon response. Dengue virus infection in human monocytic THP-1 cells led to overexpression of Hsp70, which also acts as a chaperone. The functional role of Hsp70 in dengue virus multiplication was identified by downregulating the Hsp70 gene with its specific siRNA duplexes, which led to a decrease in viral RNA copy numbers in cellular supernatants and intracellular viral load. It also resulted in an increased IFN-α level, which mediates its antiviral effect through double-stranded RNA-induced protein kinase-PKR. Collectively these results suggest that an increased level of Hsp70 expression in dengue-virus-infected THP-1 cells assists in viral replication by escaping the antiviral effect of type 1 interferon.