Immunotherapeutic modification of Escherichia coli peritonitis and bacteremia by Tinospora cordifolia.UM Thatte, MR Kulkarni, SA Dahanukar
Department of Pharmacology, Seth G.S. Medical College, Parel, Bombay.
We present here the protective effects of an Indian medicinal plant Tinospora cordifolia as compared to gentamicin in E. Coli induced peritonitis. Pretreatment with tinospora cordifolia or gentamicin reduced mortality in mice injected with 1 x 10(8) E. coli intraperitoneally from 100% in controls to 17.8% and 11.1% respectively. This was associated with significantly improved bacterial clearance as well as improved phagocytic and intracellular bactericidal capacities of neutrophils in the Tinospora cordifolia treated group. In the gentamicin treated mice although bacterial clearance was rapid, polymorph phagocytosis was depressed. Tinospora cordifolia did not possess in vitro bactericidal activity. The results demonstrate that a "prohost approach" may be beneficial in the therapy of peritonitis.
Keywords: Animal, Bacteremia, immunology,therapy,Colony Count, Microbial, Comparative Study, Escherichia coli Infections, immunology,therapy,Female, Gentamicins, therapeutic use,Immunotherapy, methods,India, Male, Mice, Peritonitis, immunology,therapy,Phagocytosis, drug effects,immunology,Plant Extracts, therapeutic use,
Intra-abdominal sepsis continues to be a major cause of morbidity and mortality following trauma and abdominal surgery for bowel perforations. Treatment of this condition has till now been focused on appropriate surgery, supplemented with antimicrobial agents and good nutritional support. An important factor, which influences recovery from any infective process is the status of the host's defence mechanism. The approach of fortifying the cellular immune functions to increase resistance against infections has only recently been recognised. Several substances, such as glucans, C. parvum, BCG and levamisol have been reported to increase resistance to infection by augmenting the immune response.
Tinospora cordifolia is a plant prescribed in Ayurveda, the Indian traditional system of Medicine as a 'Rasayana' or general tonic. It belongs to the family Memispermacea and is a large, glabrous climber with succulent, corky grooved stems. The stem is the most commonly used part and has been shown to contain a glycoside giolin, a nonglycoside bitter gilenin as well as gliosterol.
Tinospora cordifolia has been found to protect rats against mixed bacterial abdominal sepsis and mice against E.co/iinduced peritonitis, Further, cyclophosphamide myelosupperssion was also prevented by Tinospora cordifolia (TC) in mice,.
In view of the immunomodulation activities of Tinospora cordifolia, we undertook studies to initially evaluate the effect of Tinospora cordifolia upon survival of mice with E.coli peritonitis in comparison with a standard antimicrobial agent, gentamicin. Further studies to investigate the mechanism of protective effects against Ecoli sepsis were undertaken. These induced bacterial clearance studies and evaluation of polymorphonuclear functions.
Adults Swiss Albino mice, of either sex weighing between 18 and 20 gm, were used. The mice were housed in plastic cages and were fed standard diet form Hindustan Lever and allowed water ad libitum.
Tinospora cordifolia was administered as prescribed in Ayurveda . The dried, powdered stem was made into a decoction after boiling in water and administered in the dose of 100mg/kg/d by intragastric tube. Therapy was given for 15 days prior to E.coli challenge. Gentamicin (Nicholas) was given in the dose of 20 mg/mouse, intramuscular, as 2 doses, one dose 12 hours prior to and one just before E. coli challenge.
Experiment 1: Survival studies:
Sixty mice were divided into 3 groups; Group 1 served as an untreated control, Group 11 received Tinospora cordifolia (TC) and Group Ill received gentamicin.
On the test day, 1 x 10 viable E.coli were injected intraperitoneally. A clinical isolate of E.coli maintained on nutrient agar in our laboratory was used for all studies. For each experiment, E. coli was inoculated in nutrient broth for 18 hours at 35?C in a shaking water bath at 50 rpm. Bacterial cell count was adjusted to give 1 X 10 org/ml. Survival was monitored hourly during the first 24 hours and daily after that for a further 10 days.
Experiment 2: Bacterial clearance studies:
A separated set of 30 mice were divided into 3 groups (n = 10 each) as in Experiment 1. E. coli bacteremia was determined in the 3 groups by sampling tail blood at various intervals upto 16 hours (at which time the maximum mortality was recorded). The samples were serially diluted and plated on McConkey's agar in duplicate. Colony counts were determined at 24 hours after incubation at 370C.
Experiment 3: Invitro Antibacterial studies:
The in vitro antibacterial activity was tested against Escherichia More Details coli (1 x l0 org/ml)grown on nutrient agar using gentamicin as positive control. Tinospora was used in increasing strengths between 5 mg and 500 mg.
Further, the antibacterial activity of sera was obtained from mice treated with Tinospora cordifolia (100 mg/kg/d for 15 d) was also tested against the same organism. Larger plate assays were carried out using the method described by Reeves and Wise.
Experiment 4: Neutrophil function:
As the previous experiment has demonstrated that bacterial counts started reducing in the treated group after 2-3 hours, we evaluated neutrophils functions in the 3 groups (n =1 0 each) to test whether immunostimulation was the cause of bacterial clearance in the Tinospora cordifolia group 3.5 hrs after injecting E.coli into the mice on the test day, 1ml blood was withdrawn and processed by the modified method of Wilkinson to evaluate % phagocytosis (of E.coli) and % intracellular bactericidal (E.coli) capacity of neutrophils. Briefly, the blood was layered on 1 ml of 5% dextrin (MW 70,000) and erythrocytes were allowed to settle at 37?C for 1 hour. The granulocyte rich plasma was then separated. The number of neutrophils were determined. These cells were washed with Minimum Essential Medium (HiMedia), India, supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin to remove extracellular organisms. This suspension was then divided into two tubes. One set was used to assess percent phagocytosis i.e the number of neutrophils that had ingested E.coli was estimated by counting 200 cells after staining a smear of the suspension with Giemsa's stain. Phagocytic index i.e the average number of bacterial per phagocytosing was also estimated.
The other set of neutrophils was used to evaluate intracellular bactericidal capacity. The neutrophils suspension was lysed with 1 mi chilled distilled water, to release the intracellular, bacteria. The number of viable intracellular E. coli was determined by the standard pour plate method. Colony count was performed at 24 hours. The number of organisms killed per 100 neutrophils gave the % intracellular killing.
Statistical comparison between the treated and control groups was performed using Student's test. A value of p < 0.05 was considered significant. X2 test was used to calculate statistical significance for survival data. A X2 value representing 95% confidence level was considered significant.
Mice pretreated with Tinospora cordifolia (TC) (100mg/kg/d for 15d) or gentamicin showed a long-term (10 days) survival rate of 17.8% and 11.1 % respectively as compared to 0% in the control (p < 0.001).
All death occurred within the first 24 hours after challenge, the maximum mortality being recorded at 16 hours.
The rate of bacterial clearance in the 3 groups is represented in [Figure:1a] and [Figure:1b]. By the end of 8 hours the control group has severe bacteremia with average bacterial counts of 1.825 x 109 + /- X 10 9/ml of blood. By 16 hours all control mice had died. In the gentamicin treated group the counts increased upto 3 hours and then started reducing. By 8 hours the blood cultures were sterile. In the Tinospora cordifolia treated group although bacterial counts increased beyond the maximum of the gentamicin group, the peak values at 3 hours were significantly less than in untreated controls at that time. After 3 hours the bacterial count started reducing any by 16 hours blood from survivors of this group was sterile.
In vitro Antimicrobial effect:
Tinospora cordifolia exhibited no in vitro antibacterial effect at any strength. Similarly, the serum from treated animals also showed no antimicrobial effects.
Neutrophils from untreated control mice demonstrated a 34.33 + /- 3.44% phagocytosis of E.coli, 3.5 hours after challenge. As compared to this, phagocytic function of neutrophils of the Tinospora cordifolia treated group was 53.66 +/- 5.68 % (p < 0.001), stimulated, as compared to control and that in the gentamicin treated group was 19 +/- 2.75 (p < 0.001), depressed, as compared to control). The intracellular bactericidal capacity of neutrophils from the Tinospora cordifolia treated group was 52.41 +/- 5.47 % as compared to a control of 30.45 +/- 6.19% (p < 0.001).
The bactericidal capacity of neutrophils of the gentamicin was not determined effectively prevented any bacterial growth in vitro.
The present study demonstrates clearly that pretreatment with Tinospora cordifolia protect mice against mortality due to induced E.coli sepsis and this is comparable to gentamicin. This is associated with a rapid clearance of bacteremia, mediated probably through a stimulation of phagocytic and bacterial function of polymorphs. The fact that Tinospora cordifolia demonstrated no antibacterial activity in vitro further confirms that the protection afforded by Tinospora cordifolia occurred as a result of immunostimulant effects.
The use of pure bacterial cultures to induce reproducible models of intra-abdominal sepsis has been advocated by Browne and Leslie. Other substances such a glucan and zymosan have shown to protect animals against E.coli sepsis.
Tinospora cordifolia has been previously shown to produce leucocytosis with predominant neutrophila and also stimulate macrophage function,,,, in rats and mice. Acute and subacute toxicity studies (unpublished) have indicated that no significant toxicity occurred with this does regimen.
A further unique advantage of Tinospora cordifolia is that it is effective when given orally.
It appears therefore that activation of neutrophils by Tinospora cordifolia leads to a rapid bacterial clearance thus affording protection against E.coli induced peritonitis and morality.
Extrapolation of animal data to a clinical setting is not always appropriate. However, the results obtained with Tinospora cordifolia in this study coupled with its relative lack of toxicity and oral efficacy suggests that it may prove to be useful therapeutic modality in patients with abdominal sepsis.
[Figure - 1], [Figure - 2]