Studies on the Anti-Inflammatory Effect and Its Mechanisms of Sophoridine

This work is to study the anti-inflammatory effect and its mechanisms of sophoridine in vitro and in vivo. For this aim, the influences of sophoridine on several inflammatory mediators were investigated. Excessive inflammatory response in vitro model was developed by using lipopolysaccharide (LPS) to stimulate the mouse peritoneal macrophages and HL-60 cells to produce IL-6 and IL-8. Carrageenin-induced mouse paw edema model was used as inflammatory response in vivo model. MTT method, ultraviolet spectrophotometric method, and radioimmunoassay were used to measure the changes of TNFα, IL-6, PGE2, and IL-8 in in vitro cell culture supernatant or in the local inflammatory exudates. The results showed that sophoridine inhibited the production of IL-8 in in vitro cell culture supernatant and inhibited the production of TNFα, PGE2, and IL-8 in the local inflammatory exudates but had no significant effects on the production of IL-6 in vitro and in vivo. It is demonstrated that sophoridine's anti-inflammatory effect was due to its ability to inhibit the production of cytokine and inflammatory mediators.


Introduction
Inflammation is a complex physiological and pathological process accompanied by the activation of the immune system, local vascular system, and various cells within the damaged tissue. During an inflammatory process cells become activated and several mediators, such as inflammatory cytokines (TNF , IL-6, and IL-8) and inflammation media (PGE 2 , histamine), are released.
The inflammation model of carrageenin-induced edema is usually used to assess the activity of natural products in inhibiting the pathological changes associated with acute inflammation. Carrageenin can induce acute inflammation by increasing infiltration of phagocytes and oxidative stress as well as release of inflammatory mediators.
Sophoridine (SRI, C 15 H 24 N 2 O, relative molecular weight 248.36) is a quinolizidine alkaloid. SRI is also called matridine-15-one (allomatrine). It exists in overground part of Leguminous plant Sophora alopecuroides L., overground part of Euchresta japonica Benth., and root of Sophora alopecuroides Ait. It has been reported that SRI has marked pharmacological activity on anti-inflammatory [1][2][3][4], antitumor [5][6][7], and antivirus [8,9]. In our preliminary studies [10][11][12][13], we had reported some of the antiinflammatory effects and the mechanism of SRI, including the inhibitory effects on TNF production by murine peritoneal macrophages, the effect on COX in supernatant of cultured macrophages, the effect on leukotrienes (LTC 4 and LTB 4 ) biosynthesis in cells, and the effect on histamine in isolated tracheal smooth muscle and the isolated ileum of guinea pig. This work is to study the anti-inflammatory action and its mechanisms of sophoridine on cells and in vivo models continuously. For this aim, the influences of sophoridine on several inflammatory mediators IL-6 and IL-8 in LPS-stimulated mouse peritoneal macrophages and HL-60 cells and the influences of sophoridine on TNF , IL-6, IL-8, and PGE 2 in the inflammatory exudates of carrageenininduced mouse paw edema model were investigated.

Animals
Male C57BL/6 mice (6∼8 weeks) and male and female Kunming mice, body weight 18∼23 g, were obtained from Experimental Animal breeding Center of National Institutes for Food and Drug Control.

The Influences of SRI on the Production of IL-6 in LPS-Stimulated Mouse Peritoneal Macrophages: Preparation of the Mouse Peritoneal Macrophages and Calculation of the SRI Concentration Range Which Does Not Influence the Normal
Growth of Peritoneal Macrophages. The methods were the same as previously described [14]. Briefly, each male C57BL/6 mice was given an intraperitoneal injection of 1 mL 3% sodium thioglycollate solution. The animals were sacrificed after 4 days. The peritoneal macrophages were washed out with phosphate buffer solution (PBS) under a sterile environment and were washed twice with RPMI-1640 (contained 5% FBS). The cells were counted and adjusted to 2 × 10 6 cells per mL.
The 96-cell culture plate, which contained 100 L macrophage suspension (2 × 10 6 cells/mL) each well, was incubated at 37 ± 0.5 ∘ C under 5% CO 2 humidified air for 2 hours. 100 L SRI solutions (concentration range 1 × 10 −8 ∼1 × 10 −6 mol/L) were added and the plate was incubated continually for 20 hours under the same condition. 50 L MTT (2.5 mg/mL) was added each well and the plate was incubated for 4 hours. The supernatant solutions were discarded; 100 L dimethyl sulfoxide (DMSO) was added each well and mixed thoroughly. The OD values were determined at 570 nm on Microplate Reader after 5 minutes.

The Culture of Mouse Peritoneal Macrophages and
Drug Treatment. 200 L macrophage suspension (2 × 10 6 cells/mL) was added to 48-cell culture plate and incubated for 2 hours at 37 ± 0.5 ∘ C in 5% CO 2 humidified air. The nonadherent cells were washed away with PBS. 200 L RPMI-1640 culture medium (contained 10% FBS) was added to each well, and 200 L SRI solutions at different concentrations were added. After 0.5 hours of incubation, 50 L irritant agent lipopolysaccharide (LPS, 25 g/mL) was added to each well. Incubate the plate again for 24 hours before the supernatants were collected for the measurement of IL-6. [15]. 50 L supernatant was added to 96-cell culture plate, and then 50 L 7TD1 cell suspension (1.2 × 10 5 cells/mL) was added. After 48 hours incubation, MTT (5 mg/mL) 20 L was added to each well, and incubated for 4 hours. After Centrifuged under low temperature, the supernatant discarded, then DMSO 100 L was added to each well, mixed and detected the OD values at 570 nm on Microplate Reader. The decrease of the OD value in drug group indicated that the production of IL-6 in mouse peritoneal macrophages is inhibited by the drug, which was expressed in inhibition percentage. Inhibition percentage (%) = (OD LPS − OD Drug )/OD LPS × 100%. Samples were assayed in triplicate and results are reported at means (±SD).

The Influence of SRI on the Production of IL-8 in LPS-Stimulated HL-60 Cells: Calculation of the SRI Concentration Range Which does not Influence the Normal Growth of HL-60
Cells. The methods were as previously described [14]. Briefly, 100 L HL-60 cell suspension (10 5 cells/mL) was added to the 96-cell culture plate and incubated at 37 ± 0.5 ∘ C under 5% CO 2 humidified air for 2 hours. 100 L SRI solutions (concentration range 1 × 10 −8 ∼1 × 10 −6 mol/L) were added; the plate was incubated continually for 20 hours under the same condition. 50 L MTT (2.5 mg/mL) was added each well; the plate was incubated for 24 hours. The supernatant solutions were discarded after centrifuging under low temperature. 100 L dimethyl sulfoxide (DMSO) was added each well and mixed thoroughly; the OD values were determined at 570 nm on Microplate Reader after 5 minutes.

The Culture of HL-60 Cells and Drug Administration.
HL-60 cells were cultured in RPMI-1640 culture medium (containing 10% FBS) and were subcultured after one or two days. The HL-60 cells in logarithm stage were washed with D-Hunks solution and counted. The concentration of HL-60 cells was adjusted to 1 × 10 9 cell/L before use. 200 L HL-60 cell suspension was added to 48-cell culture plate. 200 L different concentrations of SRI were added and incubated for 20 minutes at 37 ± 0.5 ∘ C under 5% CO 2 humidified air. 50 L irritant agent lipopolysaccharide (LPS, 90 g/mL) was added, and the reference was added with the same amount Journal of Analytical Methods in Chemistry 3 Table 1: The results of SRI inhibited the production of IL-6 and IL-8 in LPS-stimulated mouse peritoneal macrophages and HL-60 cells ( = 9).

The Influences of SRI on Carrageenin-Induced Mouse Paw Edema and the Contents of TNF , IL-6, IL-8, and PGE 2 in the Local Inflammatory Exudates: Effect in Carrageenin-Induced
Mouse Paw Edema Model. Male and female Kunming mice (half and half, body weight 18∼23 g) were used and divided into four groups (twenty per group). Two dosages of SRI (LD 50 /5 and LD 50 /20) were given orally for 5 days (one time for each day). LD 50 of SRI is 243 mg/kg (intragastric administration). Ibuprofen was used as a positive control. 30 minutes after the last intragastric administration, 30 L of 1% carrageenan solution (solvent: 0.9% saline solution) was injected intradermally into the plantar of right hind paw. Mice were killed after 5 hours; the paws were cut at joint and weighted. The degree of swelling was expressed in the difference between the left paw weight and right paw weight. Inhibition percentage (%) = (swelling degree Model − swelling degree Drug )/swelling degree Model × 100%.

Sample Preparation for TNF , IL-6, IL-8, and PGE 2
Determination. Swollen paws of the mice were weighted, skinned, and soaked into 5 mL physiological saline at 4 ∘ C for 1 hour after cut into pieces. The swollen paw pieces were discarded and the soak solution was centrifuged. The supernatant was filtered with 0.22 m filter to remove bacteria before analysis.

Determination of TNF .
The methods were as previously described [10]. Briefly, 100 L RPMI-1640 culture solution (contained actinomycin D 2 g/mL) was added to 96-cell culture plate which is covered with a monolayer L929 cell. Then 100 L RPMI-1640 culture solution or 100 L supernatant obtained under "Samples for TNF , IL-6, IL-8, and PGE 2 determination" was added and incubated for 20 hours at 37 ± 0.5 ∘ C under 5% CO 2 humidified air. 50 L MTT (2.5 mg/mL) was added, incubated for 4 hours. After the supernatant discarded, 100 L DMSO was added to each well and mixed thoroughly. Then the OD values were read at 570 nm on Microplate Reader in 5 minutes. The RPMI-1640 culture solution wells were used as the reference. The activity of TNF in samples was expressed in cytotoxic percent. Cytotoxic percent (%) = (1 − OD sample /OD reference ) × 100. The inhibition percentage indicated the degree of depression on the production of TNF . Inhibition percentage (%) = (1 − cytotoxic percent sample /cytotoxic percent reference ) × 100%.

Determination of IL-6 and IL-8.
The detection methods were the same as described under IL-6 and IL-8 determination in mouse peritoneal macrophages and HL-60 cells. 2 . The methods were as described in [16]. Briefly, added 2 mL 0.1 mol/L KOH methanol solution to 0.1 mL supernatant obtained under "Samples for TNF , IL-6, IL-8 and PGE 2 determination". Then stored at 50 ∘ C for 20 minutes until isomerization reaction finished. Added Table 3: The influences of SRI on the production of TNF , IL-6, IL-8, and PGE 2 in the local inflammatory exudates of carrageenin-induced mouse paw edema model ( = 12). 3.167 ± 0.090 (2) 0.193 ± 0.011 (2) 0.210 ± 0.044 (1)  When the dosage is one fifth of LD 50 , SRI could inhibit the production of IL-6 and IL-8 in the local inflammatory exudates ( < 0.05), and SRI could significantly inhibit the production of PGE 2 in the local inflammatory exudates ( < 0.01). When the dosage is one twentieth of LD 50 , SRI could inhibit the production of PGE 2 ( < 0.05) but could not inhibit the production of IL-6 and IL-8. The results are listed in Tables 2 and 3.

Discussion
It has been reported [1] that SRI had some immunoregulation effect on mouse immunologic function. It could enhance the mouse macrophage phagocytic function and antagonize the immunosuppression induced by cyclophosphamide. Our previous work [10][11][12][13] had proved that SRI could significantly inhibit the production of TNF and PGE 2 in LPS-stimulated mouse peritoneal macrophages in a concentration dependent manner. And SRI had little effect on the biosynthesis of LTC 4 but inhibited the biosynthesis of LTB 4 obviously. In a certain range of concentration, SRI could significantly inhibit the contract of isolated guinea pigs bronchial smooth and ileum caused by histamine. Based on these preliminary results, the current work is to study the anti-inflammatory effect and its mechanism of sophoridine on cells, isolated organ, and in vivo models and to discuss the relationship between anti-inflammatory effect in vivo and inflammation media in cells.
The results showed that in a certain concentration range, SRI could significantly inhibit the production of TNF , PGE 2, and IL-8 in LPS-stimulated mouse peritoneal macrophages and HL-60 cells, and the inhibition effect was dose dependent. But the inhibition effect on the production of IL-6 was not significant. SRI markedly attenuated paw edema after carrageenin stimulation. Similarly, SRI could significantly inhibit the production of TNF , PGE 2, and IL-8 in the local inflammatory exudates in dose-dependent manner but could not inhibit the production of IL-6 in the local inflammatory exudates.
In summary, SRI showed inhibitory effects on inflammatory cytokines (TNF , IL-6, and IL-8) and inflammation media prostaglandin E 2 (PGE 2 ) both in cell culture supernatant (in vitro) and in the local inflammatory exudates (in vivo). Based on the experimental results obtained from both in vitro and in vivo models, the anti-inflammatory effect of SRI might be attributed to its ability to inhibit the production of cytokines (TNF , IL-8, and LTB 4 ) and inflammation media (PGE 2 and histamine) in the initial phase of inflammation.