Pharmacology And Pharmacodynamics Of Fengshikang, A Preparation From An Anti-arthritic Chinese Herbal Formula

1.Objectives and significanceRheumatoid arthritis(RA) is a Chronic,progressive,systemic disease,it's main clinical manifestations is Symmetry arthritis.Epidemiological investigation shows that there is about 0.5～1%Patients in the whole world.The incidence rate of RA in Chinese adults was about 0.3%.RA has a high disability rate.The patients with RA will usually bear high mental stress,and the quality of life is also significantly lower than the normal ones.RA is a muti-factorial disease.Its etiology is still not clear.Generally speaking, RA is a disease triggered on the base of genetic predisposition by external factors. Further more,there are many other factors are closely associated with R.A,including stature,body weight,sex,mental factors,climate,environment,and smoking,etc.Great advancement has also been attained in the researshes on path mechanism of RA.T-cell lineage is considered as a milestone in the way to the recognition of RA. In the current time,it is widely accepted that RA is resulted by the interactions between antigen presenting cell and CD4⁺T cell.Beside that,in the inflammation responses mediated by T cell,TCRγδT cell plays an important role.The balance disorders between Th1 cells and Th2 cells are also considered as one of the path mechanism of RA.In the process,protective Th2 cytokines can be generated by natural killer T cells.Cytokine network plays a key role in the damage of RA.Among the total, Tumor necrosis factor-α(TNF-α) and Interleukin(IL-1) are accepted as the most important one in the network.They maybe play an important role in inflammation occurred and development of synovial cells,synovial pannus emerged.Beside that, other cytokines,such as interleukin family,chemokine family and growth factor family,can directly participate the tissue damage.The indeterminateness of path mechanisms results in multitargets and blindness in the treatment of RA.Though the classic immune depressive therapy can improve the disease condition to some extent,even clinical cure can be attained,a long-term use of immunosuppressive agents may lead to adverse reactions,which have been frequently reported and studied.For example,methotrexate and cyclophosphamide cause disorders of gastrointestinal tract and hepatic damage;leflunomid(LEF) causes irregular menstruation,secondary pneumonia,scytitis,etc..At the same time,there is a period of drug tolerance when these drugs are applicated.For these reasons,the compliance of the patients with RA is still poor.Recent years researches on biologic agents' aimed at each target of treatment in cytokine network have made great progress.Entananercept,infliximab and recombinant interleukin-1 have been applicator in the clinical treatment.However,because of the high cost,potential health risks and too many contraindications,these agents can not widely use.On the contrary,it had been clearly explained that how and why arthromyodynia happened in the ancient traditional Chinese medicine(TCM) files.In recent years,the traditional theory of wind-cold-damp stasis was greatly improved and extended. Accordingly,TCM plays a more and more important role in the treatment of RA.As a result,the issues on how to fully explore the advantages and potential superiorities of TCM,and how to strengthen the research of the effective TCM formula,are the key points in the treatment of RA.FSK is a preparation from clinical experience of professor WU on the basis of Guizhishaoyaozhimu Decoction.FSK functions as clearing heat,eliminating dampness,quickening blood,freeing the network vessels,regulating vital energy and relieving pains.The formula involves in Cortex Phellodendri,Radix Acanthopanacis Bidentatae,Rhizoma Atracylodis,Rhizoma Anemarrhenae,Herba Asari,Radix Gentianae Macrophyllae,Ramulus Cinnamomi,Radix Cynanchi Paniculati,etc.We herein perform the research of FSK to study its anti-inflammatory and analgesic effects by pain and inflammatory animal models;we also perform animal experiments of FSK on Adjuvant arthritis rats,aimed to observe the effects of FSK on pathological changes of AA,and explore its basic pharmacological mechanism.2.Methods and project2.1 Effects of anti-inflammatory and analgesic2.1.1 Writhing experiment50 mice,female,were randomly divided into 5 groups,including Blank control group,Ibuprofen group,FSK low-dose,middle-dose,high-dose groups.Mice are intragastric administration for 3 days,30 minutes after the last one,each mouse is intraperitoneal injection by 0.6%acetic acid,and the number of writhing is recorded in 20 minutes.2.1.2 Hot-plate test50 mice,female,were put on hot plate during(55±0.5)℃,and determined the time when the mice licking the foot,the time is called pain threshold.Screening the mice that pain threshold is during 10 and 30 seconds,were randomly divided into 5 groups,including Blank control group,Prednisone group,FSK low-dose, middle-dose,high-dose groups.Mice are intragastric administration for 3 days,and screening the pain threshold at 30min,60min,and 90min.2.1.3 Ear swelling experiment50 mice,male,were randomly divided into 5 groups,including Blank control group,Prednisone group,FSK low-dose,middle-dose,high-dose groups.Mice are intragastric administration for 3 days,30 minutes after the last one,15μl xylene is spread evenly on right ear,after 15 minutes,mice are to put to death,the ears are cut, draw the materials from the same place of two ears by hole puncher,then weighing in electronic balance and calculate it.2.1.4 Permeability test of capillary vessel50 mice,female,were randomly divided into 5 groups,including Blank control group,Prednisone group,FSK low-dose,middle-dose,high-dose groups.Mice are intragastric administration for 3 days,30 minutes after the last one,caudal vein injection by 0.5%Evans Blue solution,and immediately intraperitoneal injection by 0.6%acetic acid,20 minutes later,the mice are to put to death,open the abdominal cavity,wash with 5ml Sodium Chloride,and suction the solution,centrifuge and determine the absorbance of the supernatant on extreme ultraviolet spectroscopy in 595nm.2.2 The effects of FSK on AA50 AA rats were randomly divided into 5 groups,including Blank control group, model group,LEF group,FSK low-dose,high-dose groups.On the day after the initial immunity,rats are intragastric administration for 30 days.During the treatment the arthritis score and the body weight of the rats were observed.All the knee joints and ankle joints of hind limbs were sampled after 30 days.Immunohistochemical observation and scoring was preformed. 2.3 The primary pharmacological mechanisms of FSK2.3.1 The effects of FSK on the T cell semi-group in periphery bloodTake 100μl anticoagulated blood,add the FITC-CD3,APC-CD4,PE-CD8a in order and incubation,then add the hemolysin,wash the cell by PBS,suspension the cell and detected by flow cytometer.2.3.2 The effects of FSK on the abundance of TNF-αand IL-1Enzyme linked immunosorbent assay was performed to detect serum TNF-αof AA rats,and the mean concentration of the five groups was compared and analyzed. At the same time,IL-1 in joint tissue homogenate of AA rats is detected.3.Results3.1 effects of anti-inflammatory and analgesicIn writhing experiment,compared with the control group,the times of writhing in 20 minutes are less in FSK low-dose,high-dose groups(F=30.118,P=0.000),it provide that FSK has significant analgesic effects.In the hot-plate test,there is a significant difference of the pain threshold in different time and different groups(F=19.401,P=0.000;F=5.250,P=0.001).Compared with the control group,the pain threshold are to extend in FSK low-dose,high-dose groups(P=0.013).In xylene-induced ear edema experiment,the ear swelling in FSK groups and Prednisone group was significantly lighter than the control group.And in permeability test of capillary vessel,the formula could significantly restrain the permeability of capillary vessel.FSK contributes to control the acute inflammation of adjuvant arthritis model in rats.3.2 The depressant effect of FSK on AAAfter the initial immunity,there is a significantly difference of the paw edema and the clinical score in different time and different groups,both the changes is the accordant.The mean of the paw edema and the clinical score in the model group shows significantly high.However,the one of LEF groups and FSK group shows down from the 13^th and 16^th.Observation of pathological changes showed that the pathological score of LEF groups and FSK group were significantly lower than that of the model group(F=5.121,P=0.005).It can prove that FSK has a role in controlling the acute inflammation.3.3 The primary pharmacological mechanisms of FSKThe content of CD₄⁺T cell and CD4⁺/CD8⁺ is significantly different in the model group and other medication administration group(F=5.840,P=0.002;F=12.233,P= 0.014);The content of CD₈⁺T cell is the same in the model group and other medication administration group(F=2.733,P=0.052).In FSK group and LEF group, the content of CD₄⁺T cell and CD4⁺/CD8⁺ is lower than the model groups and the content of CD₈⁺T cell is higher than the model group.The abundance of serum TNF-αand IL-1 in joint tissue homogenate of AA rats were significantly lower than that in the model group(F=2.698,P=0.044;F=11.327,P=0.000).4.Conclusion4.1 FSK has significant anti-inflammatory and analgesic effects on acute inflammation.It can extend the pain threshold,contributes to control the ear swelling and the permeability of capillary vessel.All of these can make clear that FSK has significant anti-inflammatory and analgesic effects.4.2 FSK contributes to control the acute inflammation of AA rats.FSK can suppress the pathological changes in the procession stage of AA,including synovial hyperplasia and fibrosis,inflammatory cell infiltration,the destruction of cartilages and bones.4.3 The probable pharmacological mechanisms of FSK on AA are as following:FSK can regulate the turbulence of periphery abnormal T cell semi-group of AA rats,and it may be inferred that the suppression effect of FSK on TNF-αand IL-1.