Modulation of Kupffer cell activity by Tinospora cordifolia in liver damage.DS Nagarkatti, NN Rege, NK Desai, SA Dahanukar
Dept of Pharmocology, Seth GS Medical College, Parel, Bombay.,
Kupffer cells are major determinants of outcome of liver injury. Their activity was therefore studied in a model of chronic liver disease. The effect of Tinospora cordifolia, an indigenous agent with proven hepatoprotective activity, was evaluated on Kupffer cell function, using carbon clearance test as a parameter. Rats were divided into two major groups. In Gp I which served as normal control t1/2 of carbon was 9.48 +/- 4.14 min. GpII received horse-serum in a dose of 0.5 ml/100 gm b.w. i.p. for a period of 12 weeks and was divided into three sub-groups. In Gp IIA at the end of 12 weeks half-life of carbon was found to be significantly increased to 19.86 +/- 7.95 min (p < 0.01). Indicating suppressed Kupffer cell function in chronic liver damage. In Gp IIB treated with vehicle for 4 more weeks there was significant prolongation of half-life to 38.32 +/- 10.61 min (p < 0.01), indicating perpetuation of damage in absence of damaging agent. Whereas in Gp IIc, treated with Tinospora cordifolia t 1/2 was decreased to 14.24 7.74 min (p < .01), as compared to vehicle control indicating a significant improvement in Kupffer cell function and a trend towards normalization.
Keywords: Analysis of Variance, Animal, Carbon, pharmacokinetics,Disease Models, Animal, Female, Kupffer Cells, physiology,Liver Failure, metabolism,physiopathology,therapy,Male, Metabolic Clearance Rate, Plants, Medicinal, Rats,
Chronic liver damage in humans and in the experimental animal is characterized by deposition of fibrous tissue in liver. Prognosis of chronic liver damage goes on worsening with increase in fibrous tissue content of liver. With the advent of refined biochemical and ultra-structural methodologies, it has been proved that deposition of fibrous tissue in chronic liver damage is a result of disturbance in the regulatory network between various liver Cells. Of these cells, Kupffer cells are considered as major determinant of outcome of liver injury. Under normal circumstances, these cells keep an inhibitory control on the Ito cells, the precursor fibroblasts of the liver. A suppressed activity of Kupffer cells therefore leads to proliferation and activation of Ito Cells resulting in fibrous tissue deposition. Hence, it appears that therapy directed at activating Kupffer cells will have anti-fibrotic potential.
In our laboratory, Tinospora cordifolia, a plant belonging to the family Menispermiacae, has been shown to decrease the fibrosis in animal models of reversible and irreversible liver injuries induced by carbon tetrachloride as well as heterologous serum. This antif ibrotic effect was found to preserve the liver architecture as judged by histopathology and hydroxyproline content of liver. This plant has also been shown to increase phagocytic and killing activity of macrophages, tissue elements of RES against various bacteria and fungi induced,.
Since the drug has shown anti-fibrotic effect and also found to be an activator of macrophages, it was of interest to determine whether it can modulate the activity of Kupffer cells, tissue macrophages of liver, in chronic liver damage. Hence, the following study was undertaken.
A model of fibrosis simulating post-hepatitis cirrhosis was set up. The activity of Kupffer cells in rat fibrotic liver was determined and compared with that of normal. The effect of Tinospora cordifolia therapy on fibrosis was evaluated with respect to activity of Kupffer cells. Twenty-four rats of either sex weighing between 200-500 gms were given injections of horseserum in the dose of 0.5m1/kg b.w. i.p. twice a week for 12 weeks (Gp II). A group of 8 normal rats matching in age and sex was maintained throughout this period without any treatment (GpI). At the end of 12 weeks, Kupffer cell activity, of 8 out of 24 rats (GpIIA) was measured by determining carbon clearance and compared with that of normal (Gpl).
The remaining 16 rats were divided into two groups after 12 weeks of horse-serum injections. One group received vehicle (GpIIB) and the other was treated with Tinospora cardifolia (GpIIc), both for a period of 4 weeks. Powdered stem of Tinospora cordiofolia was mixed with water and a decoction was prepared. The dose selected was 100 mg of powdered stem/kg b.w. orally once a day. At the end of 4 weeks activity of Kupffer cells was assessed.
To study the Kupffer cell activity in the above mentioned study, carbon clearance method as described by Heller (1958) was used. Initially a series of dilutions of colloidal carbon (donated by Camlin Ink) were prepared. The optical density (O.D.) of these dilutions was measured using a spectrophotometer at a wavelength of 650nm with a 1cm path length. This procedure was repeated by adding 50 ?l of blood (collected from rats) to the various dilutions of carbon and processing the samples as mentioned below. After obtaining the optical densities of these dilutions a standard curve of concentration of carbon vs optical density was constructed (Fig 1).
Blood samples were obtained from retro-orbital sinus of all the rats under test with 50?l capillaries (0 min), following which each rat was injected colloidal carbon in a dose of 8mg/100gm b.w.i.v. through femoral vein. Serial blood samples were collected at intervals of 5, 10, 15, 20,25 min., from retro-orbital sinuses with 50?l capillaries and lysed in 4ml of sodium carbonate (0.1%) solution and O.D. of these solutions were determined. These O.D. readings were used in conjunction with standard curves to calculate the amount of carbon in the blood. The concentration -time curves were plotted from which, half-life of carbon particles was calculated using least square method. This half-life of carbon particles indicates the activity of Kupffer cells.
To compare activity & Kupffer cells from different groups GpI, Gp IIA, Gp IIB, Gp IIC statistical analysis was performed by use of analysis of variance (ANOVA). If significant differences between groups were identified by ANOVA, specific comparisons between individual group were accomplished by Student's unpaired test. Results are presented as mean + S.D.
The mean optical density - concentration curve for carbon particles is depicted in [Figure:1].
The sensitivity of this test is 2.5 ng/ml. The coefficient of correlation was 0.99. The coefficients of variation for the different concentrations of carbon are as follows: 2.81%, 2.85%, 3.55%, 3.63, 2.99%, 2.21%, 2.34% for 5, 7.5, 10, 12.5, 15, 20, 22.5, 25 respectively.
The results are shown in [Table - 1].
The half-life of four groups differed significantly by ANOVA (p < 0.01). The carbon clearance in normal rats (GpI) was 9.48 + 4.14 min. In rats receiving horse-serum for 12 weeks (GpIIA) the half-life was significantly prolonged to 19.96 + 7.65 min (p < 0.01). In the rats who were administered vehicle therapy (GpIIB) forturther period of 4 weeks following horse-serum injections the half-life was found to be prolonged further to 38.32 + 10.61 min (p < 0.01 as compared to GpIIA), whereas in rats treated with Tinospora (GpIIC) for an equivalent period the half-life of carbon was found to be 14.24 + 7.74 min (p < 0.01 as compared to GpIIB).
Present study was carried out to evaluate effect of Tinospora cordifolia on Kupffer cell activity in a model of chronic liver damage. Such model of fibrosis of liver was induced in rats by repeatedly injecting horseserum. Antibodies developed against this heterologous serum combine with antigen andensuesan inflammatory process. The sequelae of such inflammation is fibrosis.
In the present study following horse-serum injection the half-life of carbon was found to be significantly prolonging (GpII A), which in turn indicates suppressed Kupffer cell activity in these animals. Depressed activity of Kupffer cells in chronic liver damage has also been reported by Noda et al.
It was observed in the present study that even if the damaging agent is withdrawn (and rats were left untreated for next 4 wks i.e. GpII B), the activity of Kupffer cells did not improve; in fact clearance of carbon was found to be significantly reduced (as judged 3. by the prolonged half-life) as compared to GpII A. Thus further suppression of Kupffer cell activity was noticed in GpIIB, which received no therapy.
On the other hand, in GpIIC the half-life of carbon clearance was found significantly less than the untreated GpIIC and comparable to control group. This indicates that Tinospora therapy has prevented deterioration of Kupffer cell activity. This appears to be due to its macrophage activating property, which has opposed any suppressant influences on Kupffer cells.
Currently a lot of research in the field of hopatology has been directed to evaluate the relation between Kupffer cell activity and development of hepatic fibrosis. It has been proved that suppression of Kupffer cells deprives the hepatocytes of cytoprotective effect, releases inhibitory control over Ito Cells, disrupts detoxification of endotoxins and decreases capacity to secrete collagenase.
In our previous study we have confirmed the deposition of fibrous tissue both by histopathological examination and by estimation of liver hydroxyproline. On discontinuation of horse serum, perpetuation of fibrotic process was observed in damaged liver. Such a phenomenon has been reported previously. Therapy with Tinospora cordifolla has been shown to prevent the fibrous tissue deposition in the post insult period.
Findings of our present study raise a possibility that this anti-fibrotic effect of Tinospora cordifolia is mediated through activation of Kupffer cells.
[Table - 1]