| ObjectiveTo study the effect of Yishen Tongbi Decoction on arthritis and bone destruction in CIA rats,and to study the anti-inflammatory mechanism of Yishen Tongbi Decoction around the JAK3 / STAT3 pathway.Methods1.Establishment of collagen-induced arthritis model:SPF female Wistar rats were selected and weighed 100g±20g.According to the weight of the animals,they were grouped by the random number table method.Type?bovine collagen was dissolved in 0.05 mmol/L acetic acid,the final concentration was 2 mg/ml,and the mixture was mixed overnight at 4? to prepare a collagen solution.The ratio of bovine type?collagen solution to an equal volume of Freund's complete adjuvant was 1:1.Mix thoroughly and repeatedly to emulsify into a water-in-oil emulsion.The emulsion finally reaches 1 mg/ml.It is administered according to the weight of the rat,0.2 ml per 100 g of body weight,and injected subcutaneously into the needle 2 cm away from the tail root.2.Rats were randomly divided into 5 groups of 10 rats each after successful modeling,10 rats in each group,,which were Yishen Tongbi Decoction low-dose group,Yishen Tongbi Decoction middle-dose group,Yishen Tongbi Decoction high-dose group,methotrexate Group and CIA model group.According to the weight of the rats,2.5 mg/kg in methotrexate group,18 g/kg in Yishen Tongbi Decoction group,9 g/kg in Yishen Tongbi Decoction group,and 4.5g/kg in Yishen Tongbi Decoction group.Started on the 1st day of foot swelling,the MTX group was administered once a week,and Yishen Tongbi Decoction was administered once a day.The CIA model group and the blank group were administered 2 ml of saline every day.All Wistar rats had normal diet.After 5 weeks of continuous administration,the rats were sacrificed and anesthetized and their materialwas taken.3.Foot edema measurement: Use 5% picric acid to mark the ankle joints of the lower limbs of the rats,and place the lower limbs in the foot edema measuring instrument according to the marks to measure the volume value of each rat,and observe the general condition of rat hair,free movement and mental state.4.Evaluation of HE: take the 3rd toe joint of the left forelimb and right hindlimb,fix,slice,HE staining,and perform assessment of neutrophil,lymphocyte and plasma cell infiltration,synovial cell proliferation.Scoring method:(1)inflammatory cell infiltration,0 points: no infiltration;1 point:1-5 HP;2 points: 6-10 HP;3 points: 11-15 HP;4 points: formation of small abscesses(2)The degree of hyperplasia of synovial fibrous tissue was 0 points without hyperplasia;1 point of hyperplasia accounted for 1/3 HP;2 points of hyperplasia accounted for 1/2 HP;3 points of hyperplasia accounted for1 HP;4 points of hyperplasia accounted for 1 HP.(3)Articular cartilage damage,0 points without damage;1 point focal lesion <1/3;2 points 1/2 damage;3points 2/3 damage;4 points all damage.5.X-ray scores: Toe and ankle destruction of each rat was evaluated in terms of erosion and joint space narrowing(JSN).Bone erosion scoring criteria:0 points: normal;1 point: suspicious or 1 small local lesion;2 points: 2local lesions or total surface area of the lesion ? 50%;3 points: 3 or more lesions or total surface area of the lesion> 50%: 4 points: Articular surface is completely destroyed,or bone shrinkage occurs.joint space narrowing(JSN):0 points: normal;1: suspicious or a small local lesion;2: joint stenosis;3: joint stiffness or joint space widening.A total of 96 bones were eroded,and there were 100 joint spaces.Scored by the radiology doctor after reading the film.6.Synovial tissue m RNA extraction and q PCR experiments to analyze the effect of Yishen Tongbi Decoction on the expression of IL-1?,TNF-? and IL-6in synovium of CIA rats.7.Flow cytometry to determine the effect of Yishen Tongbi Decoction on the ratio of Th1/Th2 of lymph nodes in rats.8.Cytotoxicity test: CCK-8 test was used to detect the effects of different concentration groups of Yishen Tongbi Decoction(YSLF)4000ng/ml,2000ng/ ml,1000ng/ml,500ng/ml,250ng/ml on Jurkat cell proliferation9.ELISA method to detect the effects of chloroform of Yishen Tongbi Decoction on TNF-?,IFN-? cytokines secreted by Jurkat cells.10.Western blot was used to observe the effect of Yishen Tongbi Decoction chloroform on the expression levels of STAT3,JAK3 and their phosphorylated proteins in Jurkat cells.Results1.Rat foot swelling value:Compared with the normal group's foot swelling value(0.69±0.05),the value of CIA group(1.67±0.28),MTX group(1.67±0.27),Yishentongbi Decoction low-dose group(1.42 ± 0.20),Yishentongbi Decoction mid-dose group(1.52 ± 0.17),Yishentongbi Decoction high dose group(1.67±0.27)was significant(P<0.05).The peak of CIA arthritis was on the7 th day after onset,and then gradually subsided,34 days after administration,MTX group(0.95±0.11),Yishentongbi Decoction mid-dose group(0.91±0.01),and Yishentongbi Decoction hig-dose group(0.96±0.23)significantly inhibited the swelling compared with the CIA group(1.42 ± 0.25)(P<0.05),there was no significant differencebetween the Yishentongbi Decoction low-dose group(1.27±0.27)and the CIA group(1.42±0.25)(P>0.05).2.X-rays of CIA rats: 35 days after administration,X-ray manifestations of CIA rats are characterized by involvement of the distal interphalangeal joint,the proximal interphalangeal joint,and the ankle with bilateral symmetry.On the 7th day after the onset of CIA,joint tissue swelling was the most worse.On the 15 th day,the joint soft tissue swelling gradually reduced.On the 34 th day,obvious bone erosion was seen,accompanied by osteoproliferative reactions.The interarticular and proximal interphalangeal joint spaces are narrowed or widened,some ankle joint spaces disappear,and joints fuse with each other to cause bony stiffness.34 days after administration,methotrexate(114.4±10.54),Yishentongbi Decoction mid-dose(123.6±7.73),and high dose(120.7±9.91)can significantly inhibit bone erosion,compared to CIA group(175.8 ± 13.42).There were significant differences(P<0.05);methotrexate(107.5±5.56),Yishentongbi Decoction mid-dose(111.9±5.61),and high dose(110.8 ±6.32)could significantly inhibit bone destruction compared to CIA group(156.1±24.68),there is a significant difference between the two(P<0.05).2.The HE pathology showed that CIA rats had obvious inflammatory cell infiltration(3.3±0.67),compared with the normal group;CIA rats had obvioussynovial tissue hyperplasia(2.8±0.79),compared with the normal group;CIA rats had obvious cartilage destruction(3.1±0.74),compared with the normal group;the medium dose group of Yishentongbi Decoction(1.6±0.70),the high dose group of Yishentongbi Decoction(1.5±0.53)and the MTX(1.4±0.52)group can inhibit inflammatory cell infiltration in CIA rats(P <0.05).Yishen Tongbi Decoction mid-dose group(1.6±0.70),Yishentongbi Decoction high-dose group(1.0 ± 0.67)and MTX group(0.9 ± 0.74)can reduce synovial cell proliferation in CIA rats(P <0.05),Yishen Tongbi Decoction mid-dose group(1.8±0.63),Yishen Tongbi Decoctionhigh-dose group(1.4±0.52)and MTX(1.2±0.63)group could reduce cartilage destruction,compared to CIA group(P<0.05).3.X-rays of CIA rats: 35 days after administration,X-ray manifestations of CIA rats are characterized by involvement of the distal interphalangeal joint,the proximal interphalangeal joint,and the ankle with bilateral symmetry.On the 7th day after the onset of CIA,joint tissue swelling was the most worse.On the 15 th day,the joint soft tissue swelling gradually reduced.On the 34 th day,obvious bone erosion was seen,accompanied by osteoproliferative reactions.The interarticular and proximal interphalangeal joint spaces are narrowed or widened,some ankle joint spaces disappear,and joints fuse with each other to cause bony stiffness.34 days after administration,methotrexate(114.4±10.54),Yishentongbi Decoction mid-dose(123.6±7.73),and high dose(120.7±9.91)can significantly inhibit bone erosion,compared to CIA group(175.8 ± 13.42).There were significant differences(P<0.05);methotrexate(107.5±5.56),Yishentongbi Decoction mid-dose(111.9±5.61),and high dose(110.8 ±6.32)could significantly inhibit bone destruction compared to CIA group(156.1±24.68),there is a significant difference between the two(P<0.05).4.The expression levels of ?-actin in each group were relatively consistent.The m RNA expressions of IL-6(1.62±0.06),IL-1?(1.05±0.07),and TNF-?(1.33±0.03)were significantly lower in the normal group,compared to expression of IL-6(19.1±0.6),IL-1?(10.75 0.19),and TNF-?(17.19±2.02)in the CIA group(P <0.05);For expression of IL-1?,methotrexate group(3.42±0.16),Yishentongbi Decoction mid-dose group(6.26± 0.32)and Yishentongbi Decoction high-dose group(4.2±0.37)was significantly lower than that in CIA group(10.75±0.19)(P <0.05).TNF-? m RNA expression in methotrexate group(6.73±0.13),Yishen Tongbi Decoction mid-dose group(8.5±0.0),and YishenTongbi Decoction high-dose group(7.14±0.25)was significantly lower than that in CIA group(17.19±2.02)(P <0.05);IL-6 m RNA expression levels in methotrexate group(9.83 ±0.13),Yishen Tongbi Decoction mid-dose group(11.51±0.37),and Yishen Tongbi Decoction high-dose group(10.75±0.19)was significantly lower than that in the CIA group(19.1±0.6)(P <0.05).5.Lymph nodes of CIA rats were significantly enlarged.Yishentongbi Decoction groups and methotrexate could inhibit lymph node enlargement of CIA rats.The proportion of Th1 cells in CIA rats was increased(38.65 ±3.52),compared with the normal group(11.50±3.27)(P <0.05);The proportion of Th2 cells in CIA rats was decreased(1.43±0.60),compared with the normal group(31.28±5.16)(P <0.05);compared with the CIA model group,the proportion of Th1 cells in Yishen Tongbi Decoction mid-dose(23.78 5.19),high-dose group(23.22 ± 4.80),and MTX group(21.87 ± 5.47)decreased significantly(P<0.05),and there was no significant difference between the low-dose decoction group(32.68±0.83)and the CIA model group(38.65±3.21)(P> 0.05).The proportion of Th2 cells in the high-dose group(19.12 ± 3.79)and MTX group(19.63 ± 3.36)were significantly increased,compared to the CIA group(1.43±0.60)(P <0.05).6.The two concentrations of Yishen Tongbi Decoction YSLF 4000ng/ml and2000ng/ ml will lead to a decrease in cell proliferation rate,while the YSLF1000ng/ml,500ng/ml,250ng/ml group has normal proliferation rates,so these three concentration gradients were selected for experiments.7.After 24 hours of PHA stimulation of Jurkat cells,the cells secreted TNF-? and IFN-?.Compared with simple PHA-stimulated cells(202.33±11.06),YSLF low-dose group(156.33±11.37)and YSLF medium-dose(139.33±5.50)and the high-dose YSLF group(112.00±8.19)reduced the levels of TNF-?secretion(P <0.05).Compared with PHA-stimulated cells(57.00±2.65),the levels of IFN-? secreted by cells in the YSLF low-dose group(31.66±4.51),YSLF mid-dose group(24.67±2.08)),and YSLF high-dose group(23.00±2.94)decreased.The difference was statistically significant(P <0.05).8.Compared with p-JAK3/JAK3((0.07±0.02)in the blank group,p-JAK3/JAK3(0.81±0.02)in the IL-2 group protein level increased,there was a significant difference(P <0.05);Compared with p-Stat3/Stat3(0.10±0.02)in the blank group,p-Stat3/Stat3(0.83±0.10)in the IL-2 group protein level increased,there was a significant difference(P <0.05);The p-JAK3/JAK3 protein levelin Yishen Tongbi Decoction mid-dose YSLF(0.07 ± 0.01),YSLF high-dose group(0.08±0.02)and JAK3 inhibitor ZM39923(0.06±0.02)was significantly lower than that in the IL-2 group(P<0.05);YSLF mid-dose(0.14±0.02),YSLF high-dose group(0.14±0.04)and the JAK3 inhibitor ZM39923(0.18±0.032)group had a lower p-Stat3/Stat3 protein level,which was significantly different from the IL-2 group(P <0.05)?ConclusionYishen Tongbi Decoction can inhibit arthritis in CIA model rats,reduce synovial hyperplasia and cartilage damage.The mechanism may be that Yishen Tongbi Decoction reduces the inflammatory cytokines IL-1?,IL-6 and TNF-? by regulating the balance of Th1 / Th2 cells,thereby inhibiting the effect of inflammatory response.YSLF can inhibit the phosphorylation of JAK3/STAT3 signaling pathway,reduce the release of inflammatory factors TNF-? and IFN-?,and exert anti-inflammatory and immune-regulating effects. |
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