Anti-inflammatory And Analgesic Effect And Mechanism Of Venenum Bufonis Ointment To Rheumatoid Arthritis

1 ObjectsRheumatoid Arthritis (RA) is a kind &chronic inflammatory diseases that is mainly to articular lesions associated with multiple systems involvement, the incidence rate is still increasing, the market has a great demand for effective drugs for the treatment of Rheumatoid Arthritis. At present western medicine are commonly practicing first-line, second-line drags and sugar cortical hormone to anti-RA, but these drugs mostly have larger side effects and adverse reactions, their dependence are strong. So searching for a safe, effective and controllable, new type Chinese medicine used to treat Rheumatoid Arthritis and improve the symptoms has positive significance.This research make ointment use of Venenum Bufonis, and use transdermal delivery method that can non- through the liver first pass effects and gastrointestinal damage, avoiding the effects of gastrointestinal enzymes, digestive enzymes, pH and other factors. The method can increase bio-availability, can to be reached the purpose of extending the role of time and reducing the toxicity and side effects of drugs, maintaining a stable and long-lasting blood concentration, provides a higher quality of treatment for RA patients.The object of this research is to explore the anti-inflammatory and analgesic effect of Venenum Bufonis ointment to treat Rheumatoid Arthritis, and inspect the joint pathological effects , that to provide a more effective and reasonable treatment of RA, and to expand the route for the administration of the drug and enhance its efficacy.2 MethodsExperimental study of this topic was divided into three sections:2.1 Analgesic effect of Venenum Bufonis ointment2.1.1 Hot plate test: SPF 18～22g NIH mice, female, were randomly divided into control group, positive control group (Ketoprofen), 3%, 6% and 12% Venenum Bufonis ointment group, After shaving abdomen the mice were treated with the drugs only 0.1g/day for 7 days. 30,60,90 min after the last administration, let the mice touch the hot plate 50℃, controlled in 1 min, counted the reaction time for the heat when mice lick the foot, compared the difference.2.1.2 Writhing test: SPF 18～22g NIH mice, half male and half female, were randomly divided into control group, positive control group, 3%, 6% and 12% Venenum Bufonis ointment group. After shaving abdomen the mice were treated with the drugs 0.1g/day for 7 days. One hour after the last administration, intraperitoneal injection of 0.6% acetic acid saline 0.01ml/g weight into mice abdomen, observed and recorded the number of mice writhing within 30 minutes, compared the difference.2.2 Anti-inflammatory effects of Venenum Bufonis ointment2.2.1 Effect 0f Venenum Bufonis ointment on rat paw swelling induced by egg white: SPF SD rats weighing 180～220g, half male and half female, were randomly divided into control group, the positive control group, and 3%,6%, 12% Venenum Bufonis ointment group. First measured rats right hind legs toe volume with toe volume machine, then treated with drugs 1g/day on the toe for 7 days. One hour after the last administration subcutaneous injection of 0.2ml fresh egg white on each mouse right foot, measured rats right hind toe volume 1,2,3 and 4h respectively, calculated swelling degree, compared the difference.2.2.2 Effect of Venenum Bufonis ointment on mice peritoneal capillary permeability: SPF18～22g NIH mice, half male and half female, were randomly divided into control group, the positive control group,and 3%,6%, 12% Venenum Bufonis ointment group. After abdominal shaving the mice were treated with the drugs 0.ig/day for 7 days. One hour after the last administration, intravenous injection of 0.5% Evans blue 0.01ml/g weight, then intraperitoneal injection of 0.6% acetic acid saline 0.01ml/g weight into mice abdomen, 20 min later killed the mice by disjointing the neck, cut open the abdomen, washed with saline 5ml several times to collect liquid and adjust the volume to end 8ml, centrifugation, supematant at 590nm spectrophotometer measured the absorbance, compared the difference.2.2.3 Effect of Venenum Bufonis ointment on granuloma induced by tampon: SPF SD rats weighing 180～220g, half male and half female, were randomly divided into control group, the positive control group, and 3%,6%,12% Venenum Bufonis ointment group. Rats ether anesthesia, disinfection the abdomen with tincture of iodine, 75% alcohol cotton deiodinase, cut lcm long small mouth, 20±2 mg autoclave tampon were implanted on both sides of axillary skin, and then suture the skin. The day before surgery were treated with the drugs 0.1g/day for 7 days, after the last administration, killed the rats by disjointing the neck, took out the tampons, removed adipose tissue, dryed up in 60℃oven, and then less that the original weight of tampon weighing in granuloma.2.2.4 Effect of Venenum Bufonis ointment on the rat adjuvant arthritis model: SPF weight 180～220g SD rats, half male and half female, were randomly divided into control group, model group, the positive control group, 3% 6% and 12% Venenum Bufonis ointment group. Subcutaneous injection on every mouse right hind foot of Freund's complete adjuvant phenobarbital of model group, the positive control group, 3% 6% and 12% Venenum Bufonis ointment group, control rats were not taken inflammatory surgery, then each group were treated with the drugs 1g/day for 7 days.Outcom measures:a.Measurement of paw swelling degree : Measured Rat paw volume before making model. Since then measured in the right hind paw volume every other week calculated paw swelling degree, compared the difference, b.Determination of tissue fluid IL-1, TNF-α, PGE₂ : One hour after the last administration, stripping the paw joints' fur, muscles, removed their toes bone and then milled the tissue, determined levels of IL-1, TNF-α, PGE₂in the rat paw tissue fluid, c.SOD activity detection: One hour after the last administration, took out 1 ml blood from the heart of each animal, centrifuge, detected serum SOD by SOD test cartridges, d.ESR detection: One hour after the last administration, took out 1.6 ml blood from the heart of each animal to 0.4ml sodium citrate anticoagulation. Inhalation of ESR tube to "0" scale,put the tubes on the ESR shelves vertically, under static at room temperature 22℃C for 1 hour, observed RBC sank a few mm. e.Paw joint pathological observation : Let blood clearance in rats after the last administration, remove fur, muscle, tendon of toe joint and exposed joints. After removing synovial membrance of toe joints, fix them in neutral formalin, paraffin-embedded after decalcification by EDTA 2 weeks, samples were HE coloration. Observe defect depth of cartilage, hyperplasia of fibrous tissue, infiltration of inflammatory cell, ect.2.3 Skin toxicology test2.3.1 Skin acute toxicity test: general New Zealand rabbit, half male and half female, weight 2.0kg, were randomly assigned to the control group (Carbomer matrix) and the experimental group (21.6% Venenum Bufonis ointment, as the greatest concentration of soluble) : Group of integrity skin and damaged skin.Each drug 3g subjects were uniformly applied to shed skin, and fixed with non- irritation gauze and bandages. Tested for 24 hours, subjects were removed after the seventh day of 1,24,48,72 hour. Observated and recorded of animal weight, hair, eyes and mucous membrane changes, respiration, the central nervous system, limbs and other activities poisoning performance daily.2.3.2 Skin irritationtest:Animal : General-New Zealand rabbit, half male and half female, weight 2.0kg around.a.Integrity skin of local irritation test : The test uses the same side of its body around contrast, 21.6% Venenum Bufonis ointment 1g to the left and Carbomer matrix 1g to the right, and fixed with non-irritation gauze and bandages. These single-dose stimulation test smeared with warm water to wash away the residue of drugs after 24 hours, after the removal of 1h, 24h,48h,72h observed of the skin reaction; Irritation Test administration repeatedly for 7 days, again after 7 days. In addition to observe and record the daily erythema and edema of the site should also observe whether there had pigmentation, hemorrhage, rough skin or skin meager, and so on. b.Damaged skin of local irritation test: 24 hours before the test, shed will be used on both sides of rabbit spine backand then friction with fine sandpaper scratches caused partial to bleeding for the local, the skin on each side of the extent of damage should be basically the same. Complete the remaining steps with Integrity skin of local skin irritation test.2.3.3 Skin sensitization test: General-albino guinea pigs, half male and half female, weight 200g～250g, were randomly divided into control group (Carbomer matrix), the positive control group (1%2. 4-nitro-chloro-2) and the experimental group (21.6% Venenum Bufonis ointment). Allergic contact : hair faded to the left; the control group smeared Carbomer Matrix 0.2g, positive control group smeared 0.2ml of 1% DNCB, the test group smeared 21.6% Venenum Bufonis ointment 0.2g. At 7 and 14 days to repeat the same method applied once. Excited contact: 14 days after the last administration, use warm water to wash the right, control group smeared Carbomer Matrix 0.2g, the positive control group smeared 0.1% DNCB 0.2ml, the test group smeared 21.6% Venenum Bufonis ointment 0.2g. Administration bred observation of skin allergies in 6h,24h,48h,72h. Recorded the animals all the time scores of allergic reactions. The same time to observe whether animals asthma, or to stand still in shock, and other serious systemic anaphylaxis.3 Results3.1 Analgesic effect of Venenum Bufonis ointment3.1.1 Hot plate test: positive control group and 3% Venenum Bufonis ointment group reaction time were significantly higher than the control group(P＜0.05), 6% Venenum Bufonis ointment group reaction time were significantly higher than the control group(P＜0.01),and significantly higher than the positive control group(P＜0.01), 12% Venenum Bufonis ointment group reaction time were significantly higher than control group(P＜0.01), 3% and 12% Venenum Bufonis ointment group with the positive control group showed no significant difference(P＞0.05).3.1.2 Writhing test : writhing number of the positive control group were much smaller than the control group(P＜0.05), writhing number of 3%,6% and 12% Venenum Bufonis ointment were significantly lower than the control group(P＜0.01).3.2 Anti-inflammatory effects of Venenum Bufonis ointment3.2.1 Effect of Venenum Bufonis ointment on rat paw swelling induced by egg white: right paw swelling degree in rats of 6% Venenum Bufonis ointment was significantly lower than the control group(P＜0.01), right paw swelling degree in rats of the positive control group,3% and 12% Venenum Bufonis ointment were significantly lower than the control group(P＜0.05).3.2.2 Effect of Venenum Bufonis ointment on mice peritoneal capillary permeability: Abdomen liquid OD value of 3%,6% and 12% Venenum Bufonis ointment group were significantly lower than the control group(P＜0.01), positive control group compared with the control group was no significant difference(P＞0.05).3.2.3 Effect of Venenum Bufonis ointment on granuloma induced by tampon: granuioma weight of positive control group, 3% ,6% and 12%Venenum Bufonis ointment were significantly lower than control group (P＜0.01).3.2.4 Effect of Venenum Bufonis ointment on the rat adjuvant arthritis model: a.Measurement of paw swelling degree: right paw swelling degree in rats of positive control group ,3%, 6% and 12% Venenum Bufonis ointment group was significantly lower than the model group(P＜0.01) . b.Determination of tissue fluid IL-1, TNF-α, PGE₂:rat paw tissue fluid IL-1 of control group were significantly lower than the model group(P＜0.01), 3%, 6% and 12% Venenum Bufonis ointment group were significantly lower than the model group(P＜0.05), positive control compared with the model group was no significant difference (P＞0.05); rat paw tissue fluid levels of TNF-αof the control group, positive control group, 3%, 6% and 12% Venenum Bufonis ointment group were significantly lower than the model group (P＜0.01); rat paw tissue fluid PGE₂ concentration of control group were significantly lower than the model group (P＜0.01), positive control group, 3% ,6% and 12% Venenum Bufonis ointment group were significantly lower than the model group(P＜0.05), c.SOD activity detection: serum SOD activity of the control group and 3% Venenum Bufonis ointment group were significantly higher than that in the model group (P＜0.05), 6% and 12% Venenum Bufonis ointmentgroup were significantly higher than model group (P＜0.01), positive control compared with the model group was no significant difference(P＞0.05), d.ESR detection: ESR rate of the control group were significantly lower than the model group (P＜0.01), the positive control group, 3% ,6% and 12% Venenum Bufonis ointment group were significantly lower than the model group(P＜0.05). e.Paw joint pathological observation :Control group : articular cartilage surface smoothness without defects, cartilage cells evenly distributed in an orderly manner; Model group : articular cartilage surface has been severely damaged, to subchondral bone tissue, fibrous tissue hyperplasia; Positive control group: articular cartilage surface small defect,mild inflammatory cell infiltration; 3% Venenum Bufonis ointment group:the joint cartilage surface defect heavier, fibrous tissue hyperplasia and infiltration of inflammatory cells significantly; 6% Venenum Bufonis ointment group: joint cartilage surface defect gently, less inflammatory cell infiltration; and 12% Venenum Bufonis ointment group: joint cartilage surface defect gently, fibrous tissue hyperplasia and infiltration of inflammatory cells lighter.3.3 Skin toxicology test3.3.1 Skin acute toxicity test: after administration of the seven days, the animals were not reduce body weight, skin integrity, no redness, no hair removal, eyes mucosa unchanged, breathing, mentally, normal activities, no other poisoning emerged performance, no one cases of animal deaths.3.3.2 Skin irritationtest: a.Integrity skin of local irritation test: single-dose 24 hours after, 21.6% Venenum Bufonis ointment skin irritation total score was 0, no stimulation. Animal activities, appetite, faeces were normal.Skin color were normal in the hours,could not found irritating reaction. Seven consecutive days administration, skin irritation score was 0, no stimulation. Animal activities, diet, manure of normal, eyes, nose had not other abnormal secretions there. No parts were found pigmentation, no bleeding, b. Damaged skin of local irritation test: single-dose 24 hours after, 21.6% Venenum Bufonis ointment skin irritation total score was 0, no stimulation. Animal activities, appetite, faeces were normal.Skin color were normal in the hours,could not found irritating reaction. Seven consecutive days administration, skin irritation score was 0.0536, no stimulation. Animal activities, diet, manure of normal, eyes, nose had not other abnormal secretions there. No parts were found pigmentation, no bleeding.3.3.3 Skin sensitization test: 21.6% Venenum Bufonis ointment sensitized guinea pig reaction score were 0, based on the incidence of allergic formula can be calculated in the incidence of allergic 0, found Venenum Bufonis ointment sensitized skin ointment for the evaluation was non-sensitized; Test found no animal asthma, stand still or shock systemic allergic reaction.4 ConclusionThrough the hot plate and writhing tese , the three concentration of Venenum Bufonis ointment had some analgesic effect.Using egg white induced paw swelling, the 3 concentration of Venenum Bufonis ointment could reduce swelling degree, had certain anti-inflammatory effect on general inflammation. Peritoneal injection of acetic acid can increase mice capillary permeability, Venenum Bufonis ointment can inhibit this role, ease capillary liquid leakage. Venenum Bufonis ointment could markedly restrain granuloma proliferation caused by the implantation of tampons.Rat adjuvant arthritis induced by injection Freund's complete adjuvant into paw. 3 concentrations of Venenum Bufonis ointment could reduce swelling degree, had a certain anti-inflammatory effect. Venenum Bufonis ointment effectively reduced the adjuvant arthritis synovial fluid IL-1, PGE₂ and TNF-αcontent, inhibited their inflammatory role, mitigating inflammatory exudation and cartilage damage. Venenum Bufonis ointment can enhance SOD activity, and removed free radicals disproportionation alleviate inflammation injury. ESR of 90% Rheumatoid Arthritis patients accelerated, the inflammation may be caused by the elements of the activation of fibrinogen so erythrocytes settling speed. Venenum Bufonis ointment slowed down the faster pace of ESR. Paw joint pathology were observed, compared with the model group, rat paw articular cartilage of the positive control group and 6%, 12% Venenum Bufonis ointment group damaged lighter, inflammatory infiltration and fibrosis were tess serious than model group, noted Venenum Bufonis ointment can prevent rat adjuvant arthritis joints damaged by inflammatory.The biggest concentration (21.6%)of Venenum Bufonis ointment in the testing process hadn't caused animals poisoned and would not cause the animals skin irritation and allergic, note Venenum Bufonis ointment using on local skin were basic medication safe.Venenum Bufonis ointment probably had anti-inflammatory and analgesic effect and prevented articular cartilage and bone destruction through regulating the immune cytokines, inflammatory mediators.