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Indian Journal of Pharmacology, Vol. 43, No. 4, July-August, 2011, pp. 441-444 Research Article The protective effect of Rubia cordifolia against lead nitrate-induced immune response impairment and kidney oxidative damage Shweta lodi, Veena Sharma, Leena Kansal Department of Bioscience and Biotechnology, Banasthali University, Banasthali, Rajasthan, India Correspondence Address: Shweta lodi Department of Bioscience and Biotechnology, Banasthali University, Banasthali, Rajasthan India shweta.tatawat@gmail.com Date of Submission: 09-Aug-2010 Code Number: ph11117 DOI: 10.4103/0253-7613.83118 Abstract Objectives: To evaluate the in vivo antioxidant activity of the ethanolic extract of the roots of Rubia cordifolia (RC) and to study its influence on lead nitrate-induced impairment of immune responses.
Keywords: Antioxidant, immunosuppression, lead nitrate, Rubia cordifolia Introduction Lead is known to induce a broad range of physiological, biochemical, and behavioral dysfunctions in laboratory animals and humans, [1] including immunological and renal dysfunctions. [2] Lead, however, was reported to have pro-oxidant catalytic activity with respect to lipid peroxidation (LPO). Several chelating agents have been used to reduce the toxic effect of lead, but these agents also have toxic potential. Rubia cordifolia (Rubiaceae) known as "manjistha" is an important medicinal plant used for treatment of various ailments such as anti-tumor, [3] anti-inflammatory, [4] and urinary disorders. [5] In view of these evidences, it was proposed to investigate whether lead nitrate can induce immunosuppression in mice and evaluate the immunomodulatory activity of a known agent like the ethanolic extract of Rubia cordifolia (RC). [6] Therefore, the objective of this study was to evaluate the effect of RC supplementation against lead toxicity in male mice. Materials and Methods Animals Male Swiss albino mice weighing 15-30 g (2-2.5 months) were obtained from Haryana Agricultural University, Hissar, India. The Animal Ethics Committee of Banasthali University, Banasthali, India, approved the study. All experiments were conducted on adult male albino mice weighing 25-35 g (3-4 months old). They were housed in polypropylene cages in an air-conditioned room at 25.3 °C, relative humidity of 50.5%, and 12-h alternating light and dark cycles. The mice were provided with chow diet (Hindustan Lever Limited, India) and drinking water ad libitum. Experimental design Seventy-two adult male Swiss albino mice (Mus musculus L.) weighing 25-30 g (3-4 months old) were used for biochemical and immunological studies. For biochemical studies, 36 mice were divided into six groups of six mice each. For immunological parameters, 36 mice were divided into six groups of six mice each. For immunological studies, all mice were challenged twice with sheep RBC with on days 14 and 20 of the experiment. The groups for each parameter were treated by oral gavage once daily as follows. Group 1 received 1 ml distilled water; served as control Group 2 received lead nitrate (40 mg/kg body weight/ day) dissolved in distilled water Groups 3 and 4 received the ethanolic extract of RC (RC) at a dose of 50 and 100 mg/kg body weight/day, respectively Groups 5 and 6 received lead nitrate (40 mg/kg body weight/ day) along with ethanolic RC (50 and 100 mg/kg body weight/day), respectively The dose for lead nitrate was decided on the basis of experiments conducted earlier and the concentration of lead nitrate used in the experiment was 1/56 of the LD 50 . [7] The plant doses were decided on the basis of experiments conducted in our own laboratory and the published reports. [8] After the administration of the last dose, the animals were given rest overnight and were sacrificed under light ether anesthesia the next day. The kidney was excised, cleaned, and washed with ice cold saline (pH 7.4), blotted, and used for biochemical assays. Serum was collected for the determination of antibody titer level from immunized mice. The spleen of immunized mice was excised and utilized for plaque forming the cell count (PFC) assay. Macrophage was collected from peritoneal cavity of immunized mice by using ice cold saline. Preparation of extract About 200 g of powdered roots (dry) were extracted with ethanol (95%) using soxhlet apparatus for 4-6 h. Alcohol removal was carried out in vacuum oven (45 °C) afforded a semi-solid mass with a yield of 9%. Chemicals Lead nitrate was purchased from Central Drug House (India). All other chemicals used were of analytical grade and obtained from Sisco Research Laboratories (India), Qualigens (India/Germany), SD Fine Chemicals (India), HIMEDIA (India), and Central Drug House (India). The immune function was assessed using macrophage yield, viability of macrophage, phagocytic index, serum immunoglobulin level, and plaque-forming cell count (PFC) in mice, whereas the oxidative stress was assessed by estimating LPO, GSH content, and the activities of superoxide dismutase (SOD) and catalase (CAT). Lipid peroxidation LPO was estimated colorimetrically by measuring malondialdehyde (MDA) as described by Nwanjo and Ojiako. [9] Lipid peroxides are produced by auto-oxidation of unsaturated fatty acids. TBA test detects only free MDA and measures the amount of free MDA in the peroxidizing lipid system. MDA reacts with thiobarbituric acid (TBA) to generate a colored product, which can be measured spectrophotometrically. The molar extinction coefficient of MDA-TBA product is 1.54 × 10 5 l/mol/cm at 535 nm. The LPO product (malondialdehyde) was expressed as nmole of MDA formed/g of tissue. Superoxide dismutase SOD activity was measured by the method suggested by Marklund and Marklund. [10] This method utilizes the inhibition of auto-oxidation of pyrogallol by SOD. The enzyme activity was expressed as unit/ml of tissue extract. Pyrogallol + O 2 --------- auto-oxidation -------- O 2 + oxidation product Catalase CAT activity in tissues was assayed as described by Aebi. [11] The CAT activity assay involves the CAT-induced decomposition of hydrogen peroxide into water and oxygen. The rate of disintegration of hydrogen peroxide into water and oxygen is proportional to the concentration of CAT. A CAT containing sample is incubated in a known amount of hydrogen peroxide. A decrease in optical density is measured at 240 nm for 60 s. The molar extinction coefficient of 43.6 M/cm is used to determine CAT activity. CAT activity was expressed as μmoles of H 2 O 2 degraded/min/mg protein. Glutathione GSH was determined by the method of Ellman. [12] The assay is based on the reduction of 5, 5?-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman′s reagent) by SH group of glutathione to form 2-nitro-S-mercaptobenzoic acid per mole of glutathione. The product is measured spectrophotometrically at 412 nm. GSH concentration was measured by using the drawn standard curve and expressed as mg/g tissue. Immunological parameters Phagocytosis was performed according to the method of Boyum. [13] For testing the viability of macrophages, 1 ml of 0.1% trypan blue was added to an equal volume of macrophage suspension. The percentage of viable cells was calculated by using the following formula:
For the evaluation of phagocytic index, macrophages were incubated with the dead E. coli culture. The number of E. coli associated with macrophages was then counted under the microscope,
The concentration of antibody in serum was determined by using GeNei TM Antibody Capture ELISA Kit (Cat No. KT51). The PFC assay was done according to the method described by Jerne and Nordin. [14] Statistical analysis The data was expressed as mean + SEM and analyzed using the Statistical Package for Social Science program (S.P.S.S. 11). For comparison between different experimental groups, one way analysis of variance (ANOVA) was used followed by post hoc Tukey′s test. The level of significance between groups was set at P<0.05. Results Renal oxidative stress and antioxidant defense related parameters Treatment with lead nitrate significantly (P<0.001) increased LPO and decreased CAT as compared to the control group [Table - 1]. However, the SOD activity was decreased by 39.81%. The ethanolic extract of RC showed a moderate effect on LPO, SOD, CAT, and GSH. Administration of lead nitrate along with RC at 50 mg and 100 mg/kg body weight significantly (P<0.001) decreased TBA reactive products as compared to the lead nitrate-exposed group. Moreover, a significant (P<0.001) elevation in the CAT and GSH content was also observed in comparison to lead nitrate-treated animals. SOD activity also increased when treated with RC as compared to the lead nitrate control group. Immunological alterations Lead nitrate treatment showed significant reduction (P<0.001) in macrophage yield, viability of macrophage, phagocyte index, serum immunoglobulin level, and PFC as compared to control group. Ethanolic extract of roots of RC had no effect on macrophage yield, viability of macrophage, phagocytic index and PFC. Whereas high dose of RC increased phagocytic index, PFC and serum Ig levels. The macrophage yield and viability of macrophages and phagocytic index significantly increased (P<0.001 and P<0.05) when treated with lead and ethanolic extract of RC at 50 mg and 100 mg/ kg body weight respectively [Table 2]. The combination treatment also raised PFC significantly (P<0.001), when compared with lead nitrate administered group. Discussion The present study observed a marked alteration in the peroxidative process following lead nitrate exposure. There was increase in LPO and decrease in SOD, CAT, and GSH in renal tissue of mice treated with lead nitrate. Similar results have been reported by us. [2],[15],[16],[17],[18] Lead treatment enhances LPO in renal tissue of mice that is directly related to free radical mediated toxicity. The target of oxidative damage is usually biomolecules such as nucleic acid, proteins, and lipids. [19] Lead absorbed to blood and tissues produces highly reactive species such as superoxide radical ( 2 O−·), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (·OH) and lipid peroxides. The measurement of LPO is a deleterious process that involves continuous fragmentation of membrane lipids and very often, heavy metal toxicity has been associated with this process. The lipid peroxides, in turn, gets degraded into a variety of products, including alkanals, hydroxyl alkanals, ketones, alkenes, etc. All of these products inactivate cell constituents by oxidation or cause oxidative stress by undergoing radical chain reaction. Such changes may lead to disintegration of membrane structure and irreversible cell damage. However, endogenous enzyme such as SOD and CAT can protect against peroxidation damage to the biomembranes. Both the enzymes are important against oxygen metabolism. [20] The first line of defense against superoxide free radicals is the enzyme known as "Superoxide dismutase" or (SOD), that is considered the most effective antioxidant. Depletion of SOD activity was observed during lead exposure. Decrease in SOD activity can be attributed to an enhanced superoxide production during lead metabolism. In the present study superoxide radical also inhibits the activity of CAT. GSH is an important cellular antioxidant defense system against free radical overproduction and decreasing of its cellular concentration impairs cellular defense against oxidative stress. [15] The depletion of GSH content in the present work promotes generation of ROS and oxidative stress with cascade of effects thereby affecting function as well as structural integrity of cell and organelle membrane. Thus it can be said that lead disturbs pro-and anti- oxidative balance in the tissues causing oxidative stress explaining its toxic nature. The effects of antioxidant clearly demonstrate the generation of reactive oxygen species by metal. However, administration of RC extract with lead nitrate significantly prevented the influence of lead on antioxidative system . It decreased LPO and concomitantly increased the activities of SOD, CAT and GSH levels in renal tissue. These results clearly demonstrate the anti peroxidative role of the plant. The in vivo protection by RC extract against lead induced oxidative damage may be because of its free radical scavenging potential. It could also be because of direct scavenging/neutralization of the free radical or induction of the endogenous antioxidant enzymes such as CAT and SOD. RC extract possesses polyhydroxyl substituted anthraqunions that may be responsible for protective properties. [21] Besides, RC extract significantly chelates iron, [21] maintains the level of GSH, contains vitamin C [22] and also scavenges the hydroxyl radicals. Thus present study demonstrates the free radical scavenging antioxidant properties of the ethanolic root extract of the plant. A limited amount of data suggest that the biochemical and molecular mechanisms of lead toxicity involve the induction of oxidative stress in target cells, partly via the activation of reactive oxygen species (ROS), followed by DNA damage and apoptosis. Generation of ROS appears to be central for the immunotoxic effects of lead. [23] The present study showed that lead nitrate led to decrease in cell mediated immune response determined in terms of viability of macrophage and phagocytic index. Impairment of phagocytosis by lead may be due to the depletion of glutathione. Systemic administration of lead nitrate also revealed a significant decrease in serum immunoglobulin level and plaque formation, relative to control group, indicating that lead nitrate suppresses the mice humoral immune response. These results are in accordance with published report that showed lead administration can have inhibiting effect on the production of antibodies, possibly due to a direct toxicity on the B-lymphocytes. [24] Mice treated with RC alone had no significant effect on immune system. However, combination treatment significantly improved the altered immune responses suggesting immune-stimulating properties and protection against lead nitrate induced oxidative damage. Similar results have been observed and the effects were comparable to that of vitamin E and C. [25] Hence, the study concludes that immunomodulatory effect of RC may be subsequent to the its antioxidant activity. [25] References
Copyright 2011 - Indian Journal of Pharmacology The following images related to this document are available:Photo images[ph11117t1.jpg] [ph11117t2.jpg] |
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