Ipomoea obscura ameliorates cyclophosphamide-induced toxicity by modulating the immune system and levels of proinflammatory cytokine and GSH

Ipomoea obscura L. is a widely used medicinal plant. The objective of this study was to investigate its protective activity against cyclophosphamide (CTX)-induced toxicity in mouse models. Swiss albino mice were treated intraperitoneally with CTX (25 mg/kg body weight) along with I. obscura extract (10 mg/kg body weight) for 10 days. Extract significantly reduced myelosuppression caused by CTX and improved the relative organ weight, total white blood cell count, and bone marrow cellularity. The elevated levels of parameters related to pathophysiology of the liver, namely glutamate pyruvate transaminase, alkaline phosphatase, and lipid peroxidation, were significantly reduced by extract treatment. Reduction of liver and intestinal glutathione levels of CTX-treated animals was reversed by I. obscura. The lowered levels of cytokines, namely IFN-γ, IL-2, and granulocyte-monocyte colony-stimulating factor after CTX treatment were found to be increased in I. obscura treated animals. Treatment with I. obscura could also decrease the level of proinflammatory cytokine TNF-α. The data suggested that I. obscura can act as a potent chemoprotective agent and can be used as an adjuvant in chemotherapeutic applications.     

Hypoglycemic effect of methanol extract of Phyllanthus amarus Schum & Thonn on alloxan induced diabetes mellitus in rats and its relation with antioxidant potential

Methanolic extract of P. amarus was found to have potential anti-oxidant activity as it could inhibit lipid peroxidation, and scavenge hydroxyl and superoxide radicals in vitro. The amount required for 50% inhibition of lipid peroxide formation was 104 microg/ml and the concentrations needed to scavenge hydroxyl and superoxide radicals were 117 and 19 microg/ml respectively. The extract was found to reduce the blood sugar in alloxan diabetic rats at 4th hr by 6% at a dose level of 200 mg/kg body wt and 18.7% at a concentration of 1000 mg/kg body wt. Continued administration of the extract for 15 days produced significant (P < 0.001) reduction in blood sugar. On 18th day after alloxan administration values were almost similar to normal in the group taking 1000 mg/kg body wt.     

Ganoderma spore lipid protects mouse bone marrow mesenchymal stem cells and hematopoiesis from the cytotoxicity of the chemotherapeutic agent

Cancer chemotherapeutic agents are frequently toxic to bone marrow and impair bone marrow functions. It is unclear whether ganoderma spore lipid (GSL) can protect bone marrow cells from the cytotoxicity of chemotherapy. To investigate the protective effects of GSL on bone marrow mesenchymal stem cells (MSCs) and hematopoiesis, we examined the effects of GSL on MSCs in vitro and hematopoiesis in vivo after treatment with the chemotherapeutic agent cyclophosphamide. MSCs and peripheral blood cells were isolated and counted from the bone marrow of normal mice were pre-treated with GSL before CTX treatment or co-treated with GSL and CTX, followed by examining the changes in phenotype, morphology, proliferation, apoptosis, and differentiation potentials. The results showed that GSL could reduce the CTX-induced changes in the phenotype of MSCs and maintain the elongated fibroblast-like morphology. MTT and annexin V/propidium iodide (PI) analyses found that GSL pre-treatment and co-treatment increased the proliferation and decreased the apoptosis in CTX-treated MSCs. Furthermore, GSL improved the osteogenic and adipogenic differentiation potentials of CTX-treated MSCs. In vivo, GSL treatment increased the number of peripheral blood cells including white blood cells (WBC) and platelets (PLT) in the CTX-treated mice and enhanced the in vitro formation of hematopoietic lineage colonies (erythrocyte colony forming unit, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte macrophage colony-forming units, CFU-GM) from bone marrow cells in these mice. These findings suggest GSL could protect MSCs and hematopoiesis from the cytotoxicity of CTX and might become an effective adjuvant to attenuate side effects of chemotherapy during cancer treatment.     

Immunomodulatory activity of Sulforaphane, a naturally occurring isothiocyanate from broccoli (Brassica oleracea)

The effect of Sulforaphane on the immune system was studied using BALB/c mice. Intraperitoneal administration of five doses of Sulforaphane (500 microg/dose/animal/day) was found to enhance the total WBC count (12,950 cells/mm3) on 9th day. Bone marrow cellularity (23 x 10(6) cells/femur) and number of alpha-esterase positive cells (1346.66/4000 cells) were also increased by the administration of Sulforaphane. Treatment with Sulforaphane along with the antigen, sheep red blood cells (SRBC), produced an enhancement in the circulating antibody titre and the number of plaque forming cells (PFC) in the spleen. Maximum number of PFC (315.83 PFC/10(6) spleen cells) was obtained on the 6th day. Administration of Sulforaphane also showed an enhancement in the phagocytic activity of peritoneal macrophages. Moreover administration of Sulforaphane significantly reduced the elevated level of TNF-alpha production by LPS stimulated macrophages. These results indicate the immunomodulatory activity of Sulforaphane.     

Immunopotentiating activity of abrin,a lectin from Abrus precatorius Linn

A non-toxic dose of abrin, (1.25 microg/kg body wt) consecutively for five days in normal mice stimulated specific humoral responses. A noticeable increase was observed in total leucocyte count, lymphocytosis, weights of spleen and thymus, circulating antibody titre, antibody forming cells, bone marrow cellularity and alpha-esterase positive bone marrow cells. The results suggest that abrin can potentiate the humoral immune response of the host.     

Augmentation of myelopoiesis in a murine host bearing a T cell lymphoma following in vivo administration of proton pump inhibitor pantoprazole

Proton pump inhibitors (PPI) are being proposed as potent antitumor agents, owing to their ability to specifically induce tumor cell death by reversing H⁺ ion homeostasis. As tumor growth induces myelosuppression in tumor-bearing hosts, it remains unclear if PPI can also modulate tumor-induced myelosuppression. Thus, we studied the effect of in vivo administration of pantoprazole (PPZ), a PPI, on myelopoiesis in a murine model of a transplantable T cell lymphoma, designated as Dalton’s lymphoma (DL). Intraperitoneal administration of PPZ to tumor-bearing mice resulted in an enhanced bone marrow cellularity, inhibited induction of apoptosis and augmented bone marrow cell (BMC) survival. BMC of PPZ-administered tumor-bearing mice showed elevated number of F4/80 positive cells, augmented colony forming ability and differentiation in bone marrow-derived macrophages (BMDM) with higher expression of F4/80 and CD11c markers. This study also presents evidences to indicate that PPZ-dependent augmentation of myelopoiesis in the tumor-bearing host is dependent on an enhanced expression of M-CSF and receptors for M-CSF & GM-CSF in BMC, along with a modulation in the expression of cell survival regulatory molecules PUMA, Bcl2, p53 and caspase-activated DNase (CAD). BMDM obtained from PPZ-administered tumor-bearing mice also showed an augmented expression of TLR-2, tumoricidal activity, production of NO and monokines: IL-1, IL-6 & TNF-α. The study discusses the possible mechanisms underlying PPZ-dependent augmentation of myelopoiesis. Taken together, the present study proposes that a PPZ-dependent alleviation of tumor-induced myelosuppression could contribute to an augmented myelopoiesis.     

Glucan effect on the survival of mice after radiation exposure

The effect of glucan (beta-1,3-polyglucose) was investigated on the radiation-induced damage to the system of non-specific immunity in mice. A positive influence of glucan administered before exposure to a dose of 200 R was observed on the following parameters of postradiation regeneration: while blood cell count, due mainly to increased granulocyte count, increased per cent of peroxidase-positive cells in bone marrow, increased mass and cellularity of the spleen, in relation to the group of the animals not receiving the preparation. Administration of glucan 5 days before exposure to 650 R of X-rays prolonged the mean survival time. Administration of the preparation after exposure to radiation had no significant effect on the survival time of mice.     

The effect of dextran sulphate on CFU-S and nucleic acids in blood and haemopoietic tissues in irradiated mice.

The effect of synthetic polyanion dextran sulphate on the development and recovery of radiation-induced haemopoietic damage in mice was investigated. Dextran sulphate (mol. wt. 500,000 D) in the dose of 40 mg.kg-1 of body weight was injected i.p. 3 days before single total body irradiation with a dose of 7.8 Gy gamma-rays. The animals were examined from hour 6 to day 26 after irradiation, i.e. from hour 78 to day 29 after DS-treatment. In irradiated mice DS-pretreatment showed some positive effect on the CFU-S number in bone marrow (less in spleen and blood), bone marrow cellularity, attenuated the radiation-induced changes of erythrocytes (number, MCV) and of RNA concentration in blood. The changes of other parameters (spleen cellularity, liver CFU-S, leukocyte count and DNA concentration in blood) were the same as in unprotected animals. In conclusion, we can say that DS-pretreatment had a beneficial effect on the recovery of radiation-induced damage of erythropoiesis but not on granulopoiesis or lymphopoiesis.     

Effect of herbal preparation, brahma rasayana, in amelioration of radiation induced damage

Oral administration of brahma rasayana (BR; 10 and 50 mg/dose/animal) for 15 days increased significantly total leukocyte count and percentage of polymorphonuclear cells in irradiated mice. Bone marrow cellularity and alpha-esterase positive cells also increased significantly in radiation-treated animals after BR administration. Number of nodular colonies on the surface of spleen on day seven increased significantly in lethally irradiated recipients receiving bone marrow cells from animals treated with BR. Oral administration of BR also enhanced in serum level of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor(GM-CSF) in normal and irradiated mice. These results indicated that proliferation of stem cells induced by BR in irradiated mice may be related to its stimulation of cytokine production.     

Effect of liposomal muramyl tripeptide phosphatidylethanolamine and indomethacin on hematopoietic recovery in irradiated mice

The effects of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE/MLV, radioprotective immunomodulator; 10 mg/kg) and indomethacin (INDO, inhibitor of prostaglandin production; 2 mg/kg) on post-irradiation recovery of hematopoietic functions in mice were investigated. Two agents with distinct radioprotective mechanisms were administered alone or in combination 24 h and 3 h before exposure to 7 Gy (60)Co radiation. In the post-irradiation period (3-14 days) combined pre-treatment of mice accelerated recovery of bone marrow cellularity, weight of spleen and myelopoietic and erythropoietic activity in both hematopoietic organs, compared to treatment with MTP-PE/MLV or indomethacin alone. In the peripheral blood, improved radioprotective effects of combined drug administration were found in the recovery of reticulocytes and platelet count. No further significant differences in the recovery of leukocyte count were observed in the examined groups until post-irradiation day 14. Within the first 3-6 post-irradiation days, the bone marrow and peripheral blood smears of mice pre-treated with indomethacin alone or its combination with MTP-PE/MLV more frequently featured blast cells and large cells with abundant cytoplasm which could be considered the hematopoietic stem cells.     

Effect of AC II, a herbal formulation on radiation-induced immunosuppression in mice

A single dose of 6 Gy irradiation significantly reduced the total WBC count while in herbal formulation (AC II) treated groups it was found to be significantly increased. Similarly bone marrow cellularity and alpha-esterase positive cells, which were lowered by radiation, were partly restored in AC II treated groups. The data indicate that AC II can overcome the immunosuppression produced by irradiation.     

超强静磁场联合环磷酰胺对S180荷瘤小鼠抗肿瘤和相关生理指标的影响

实验探讨了超强静磁场(ultra strong static magnetic field,USMF)联合环磷酰胺(cyclophosphamide,CTX)连续处理,对S180荷瘤小鼠抗肿瘤、抗氧化、免疫及骨髓抑制等方面的影响。对S180肉瘤小鼠分组处理后,剥取肉瘤组织称重并进行病理检验。检测过氧化氢酶、超氧化物歧化酶和谷胱甘肽过氧化物酶的活力、总抗氧化能力,以及肝脏和肾脏中丙二醛的含量、脾脏和胸腺指数、脾脏淋巴细胞转化率、外周血中的白细胞数目和骨髓细胞的DNA含量。腹水瘤小鼠同样处理后正常饲养,记录生存时间。结果发现,USMF+CTX组的抑瘤率(72.5%)比CTX组(51.5%)提高了40.8%,生命延长率提高了1.5倍,抗氧化和免疫能力也有一定程度的增强。表明USMF结合CTX,可以协同性抑制S180荷瘤小鼠肿瘤的生长,并减轻CTX对小鼠的副作用。     

Single administration of low dose cyclophosphamide augments the antitumor effect of dendritic cell vaccine

Single administration of low dose cyclophosphamide (CTX) was previously reported to enhance the antitumor efficacy of immunotherapies. To investigate the possible mechanisms for this effect, we examined whether a single administration of low dose CTX could augment the immunogenicity of dendritic cell (DC) vaccines. Fifty milligrams per kilogram body weight dose of CTX was administrated intraperitoneally to mice after B16 melanoma or C26 colon carcinoma tumor models were established, DC vaccine generated from mouse bone marrow and pulsed with B16 or C26 tumor cells lysates were vaccinated 4 days later. CTX treatment potentiated the antitumor effects of the DC vaccine, and increased the proportion of IFN-γ secreting lymphocytes in spleens. Furthermore, a significantly reduced proportion of CD4+CD25+FoxP3+ regulatory T (T_reg) cells was detected by flow cytometry in spleen lymphocytes from tumor-bearing mice treated with CTX. Thus, a single administration of low dose CTX could augment antitumor immune responses of DC vaccine by reducing the proportion of CD4+CD25+FoxP3+ T_reg cells in tumor-bearing mice. Our results suggested a possible mechanism of CTX-induced immunopotentiation and provided a strategy of immunotherapy combining a low dose CTX with DC vaccine. J.-Y. Liu and Y. Wu contributed equally to this work.     

Ginsenoside Rg1 improves bone marrow haematopoietic activity via extramedullary haematopoiesis of the spleen

Cyclophosphamide (CY) is a chemotherapeutic agent used for cancer and immunological diseases. It induces cytotoxicity of bone marrow and causes myelosuppression and extramedullary haematopoiesis (EMH) in treated patients. EMH is characterized with the emergence of multipotent haematopoietic progenitors most likely in the spleen and liver. Previous studies indicated that a Chinese medicine, ginsenoside Rg1, confers a significant effect to elevate the number of lineage (Lin⁻) Sca-1⁺ c-Kit⁺ haematopoietic stem and progenitor cells (HSPCs) and restore the function of bone marrow in CY-treated myelosuppressed mice. However, whether the amelioration of bone marrow by Rg1 accompanies an alleviation of EMH in the spleen was still unknown. In our study, the cellularity and weight of the spleen were significantly reduced after Rg1 treatment in CY-treated mice. Moreover, the number of c-Kit⁺ HSPCs was significantly decreased but not as a result of apoptosis, indicating that Rg1 alleviated EMH of the spleen induced by CY. Unexpectedly, the proliferation activity of c-Kit⁺ HSPCs was only up-regulated in the spleen, but not in the bone marrow, after Rg1 treatment in CY-treated mice. We also found that a fraction of c-Kit⁺/CD45⁺ HSPCs was simultaneously increased in the circulation after Rg1 treatment. Interestingly, the effects of Rg1 on the elevation of HSPCs in bone marrow and in the peripheral blood were suppressed in CY-treated splenectomized mice. These results demonstrated that Rg1 improves myelosuppression induced by CY through its action on the proliferation of HSPCs in EMH of the spleen and migration of HSPCs from the spleen to the bone marrow.     

OK-432 stimulates primary production and activity of murine natural killer cells

Although many immunostimulants have been shown to increase the lytic activity of natural killer (NK) cells in the periphery, little is known about their effects on NK cells in the bone marrow, the primary site of NK production. In the experiments reported here, we tested OK-432, a pharmaceutical preparation of Streptococcus pyogenes, for its effects on both the primary production and lytic activity of NK cells in C57BL/6J mice. NK activity in bone marrow cells (BMC) and spleen cells (SC) was significantly increased following intravenous administration of OK-432, peaking on day 2 in BMC and on day 3 in SC. Concomitantly, there were marked changes in the cellularity in the two compartments. Bone marrow cellularity fell significantly on day 1 post-OK-432 and then gradually returned to normal, whereas spleen cellularity rose rapidly and remained elevated. As a consequence, the total NK activity (per femur or per spleen) was significantly increased at 48-96 h after administration of OK-432. The target specificity was unchanged. The phenotype of NK cells in BMC as determined by cytotoxic depletion was unchanged by OK-432, but splenic NK activity shifted to a 'less mature' phenotype, intermediate between that of normal BMC and SC. Cytokinetic studies using 3H-TdR revealed an increase in the production of NK cells in the bone marrow following administration of OK-432. Proliferating NK cells also appeared in the spleen. Whether these were recently produced NK cells from the bone marrow that still retained the ability to proliferate or mature NK cells that were stimulated into cell cycle cannot be determined from these experiments. These data are the first to directly demonstrate the modulation of the primary production of NK cells by an immunologically active drug.     

Effect of naturally occurring triterpenoids glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin on the immune system.

The effect of naturally occurring triterpenoid compounds such as glycyrrhizic acid, ursolic acid, oleanolic acid, and nomilin on the immune system was studied using Balb/c mice. Intraperitoneal treatments with 5 doses of these terpenoid compounds were found to enhance the total white blood cells (WBC) count. In ursolic acid, oleanolic acid and nomilin treated animals the maximum total WBC count was observed on the 6th day, while in glycyrrhizic acid treated animals it was observed only on the 9th day after the drug treatment. In ursolic acid, oleanolic acid and nomilin treated animals the percentage of increase in the total WBC count was to 91.48 +/- 4.6%, 135.75 +/- 6.4% and 117.33 +/- 17.9% respectively. In the glycyrrhizic acid treated animals the total WBC count was increased to 114.9 +/- 18%. Bone marrow cellularity and alpha-esterase positive cells were also enhanced by the treatment with these terpenoids. Treatment with various triterpenoids along with antigen produced an enhancement in the specific antibody titre and the number of plaque forming cells (PFC) in the spleen. Triterpenoids remarkably inhibited delayed type hypersensitivity reaction (DTH). These results indicate the immunomodulatory activity of naturally occurring triterpenoids such as glycyrrhizic acid, ursolic acid, oleanolic acid and nomilin.     

Amelioration of cyclophosphamide induced myelosuppression and oxidative stress by cinnamic acid

Cinnamic acid (C9H8O2), is a major constituent of the oriental Ayurvedic plant Cinnamomum cassia (Family: Lauraceae). This phenolic acid has been reported to possess various pharmacological properties of which its antioxidant activity is a prime one. Therefore it is rational to hypothesize that it may ameliorate myelosuppression and oxidative stress induced by cyclophosphamide, a widely used chemotherapeutic agent. Commercial cyclophosphamide, Endoxan, was administered intraperitoneally to Swiss albino mice (50mg/kg) pretreated with 15, 30 and 60mg/kg doses of cinnamic acid orally at alternate days for 15days. Cinnamic acid pre-treatment was found to reduce cyclophosphamide induced hypocellularity in the bone marrow and spleen. This recovery was also reflected in the peripheral blood count. Amelioration of hypocellularity could be correlated with the modulation of cell cycle phase distribution. Cinnamic acid pre-treatment reduced bone marrow and hepatic oxidative stress as evident by lipid peroxidation and activity assays of antioxidant enzymes such as superoxide dismutase, catalase and glutathione-S-transferase. The present study indicates that cinnamic acid pretreatment has protective influence on the myelosuppression and oxidative stress induced by cyclophosphamide. This investigation is an attempt and is the first of its kind to establish cinnamic acid as an agent whose consumption provides protection to normal cells from the toxic effects of a widely used anti-cancer drug.     

Assessment of anti-mutagenic,anti-histopathologic and antioxidant capacities of Egyptian bee pollen and propolis extracts

Bee pollen and propolis are popular, traditional health foods. The objective of the current study was to investigate the anti-mutagenic, anti-histopathologic and antioxidant effects among water extracts of Egyptian bee pollen (WEBP) and brown powder of water-soluble derivative propolis (WSDP) on cisplatin (CDDP) induced hepatic, renal, testicular and genotoxicity in male albino mice (Mus muscullus), in addition to their effects on the oxidant/antioxidant status in the tested organs. Hepatic, renal and testicular dysfunctions were evaluated histologically; while genotoxicity and cytotoxicity were evaluated by the bone marrow chromosomal aberration assay and mitotic index, respectively. Moreover, oxidative stress was explored via determination of lipid peroxidation, catalase activity and the concentration of the reduced form of glutathione. The treatment of mice with WEBP and WSDP at doses 140 and 8.4 mg/kg b. wt./day, respectively for 14 days simultaneously with CDDP (2.8 mg/kg b. wt.) resulted in significant protection. The positive control animals taken CDDP alone showed toxic histological and genetical manifestations (at P < 0.05) accompanied with an elevated content of peroxidized lipid and lowered catalase activity and glutathione concentration in the homogenate of liver, kidney and testis tissues (at P < 0.001). These toxic side effects in all tested organs were greatly ablated with a significant reduction in lipid peroxidation level and elevation in catalase activity and glutathione concentration (P < 0.001) when using both WEBP and WSDP. On the basis of the present assays, Bee pollen appears more potent in exerting an ameliorative effect and this effect was more pronounced in testis.     

Effect of dipyridamole on the interrelations of the stromal and hematopoietic tissues of the bone marrow in experimental studies]

By means of heterotopic transplantation of the bone marrow interrelations of the stromal and hemopoietic tissues of the mice bone marrow have been studied at administration of dipiridamol. Effect of the drug to the hemopoiesis is realized via stem stromal cells of the bone marrow. Under the influence of dipiridamol a focus of heterotopic hemopoiesis the osteogenic component in it is present only in 30% of cases in comparison with the control. Inhibition of the stromal component proliferation is accompanied with increasing mitotic activity of the hemopoietic elements against the background of the bone marrow cellularity decrease both in the femoral bone and in the focus of heterotopic hemopoiesis. At administration of dipiridamol a phenomenon of noneffective megakaryocytopoiesis with the intrabone marrow destruction of megakaryocytes, resulting in local release of thrombocyte growth factor, which has a compensatory character.     

Formation of a heterotopic hemopoiesis focus from the administration of a cryptococcal heteropolysaccharide to bone marrow donors and recipients

The effect of Cryptococcus luteolus (strain 228) heteropolysaccharide on heterotopic bone marrow organ formation was studied. Heteropolysaccharide (Hp) injections were initiated on the next day after bone marrow transplantation and were performed weekly for 30 days. This resulted in the increase in both the cellularity and the weight of the ossicle. The injections performed twice weekly produced the decrease of cellularity while ossicle weight did not change. Higher dose of Hp produced no changes in heterotopic bone marrow organ. Hp injections to donor mice did not result in any changes in heterotopic organ in bone marrow recipients. Thus, this kind of Hp was capable both of stimulating and inhibiting heterotopic bone marrow organ formation in relation to the dose and scheme of administration.