| BackgroundRheumatoid arthritis(RA)is a common autoimmune disease with unknown etiology and pathogenesis.The main clinical manifestation is symmetrical multi-joint involvement.Repeated and persistent attacks of RA will eventually lead to progressive destruction of the patient’s joints and even loss of self-care ability.In China,5 million people are deeply troubled by RA disease,which is also one of the important reasons for the loss of labor force in our country.At present,there is no drug that can cure RA.The main clinical drugs are non-steroidal anti-inflammatory drugs,disease-modifying anti-rheumatic drugs,and glucocorticoids.These drugs have many adverse reactions,so the degree of disease remission in patients is still far low.than expected.In recent years,molecular targeted therapy has become a research hotspot,and JAK is undoubtedly one of the most popular targets.Some small-molecule inhibitors targeting JAK kinases have been approved for the treatment of RA,but there are still some patients who do not respond well to such preparations,are resistant to these preparations,have high potential infection risks,are expensive,and have long-term efficacy.And safety needs to be further evaluated,the development of safe,cost-effective and efficient drugs is of great significance for the treatment of RA.Traditional Chinese medicine treatment is guided by the theory of traditional Chinese medicine,and has the advantages of multi-level and multi-target effects.My tutor,Professor Guangxing Chen,has been using Yishen Tongbi Decoction(YSTB)in the clinical treatment of RA for more than ten years.The previous clinical research of our group found that YSTB can improve the symptoms of joint pain and swelling in RA patients,and reduce inflammatory indicators such as ESR and CRP;at the animal and cellular levels,YSTB can reduce the acute joint response in rats and inhibit the proliferation and activation of B cells.However,the specific mechanism of action of YSTB in the treatment of RA remains to be further studied.ObjectiveThis study was based on the observation of the effect of Yishen Tongbi Decoction on the inflammatory response and bone destruction in a mouse model of collagen-induced arthritis(CIA),and the effect of Yishen Tongbi Decoction on RAW264.7 cells.In order to elucidate the effect of Yishen Tongbi Decoction on RA by regulating the JAK/STAT3/SOCS pathway.Through the observation of pharmacodynamics and the study of the mechanism of action,it is expected to bring new ideas on the pathogenesis of RA and the mechanism of prevention and treatment of traditional Chinese medicine,and to provide a scientific theoretical basis for the clinical application of Yishen Tongbi Decoction in the treatment of RA.Methods1.Animal experiments:To explore the anti-inflammatory and anti-bone destruction effects and mechanisms of Yishen Tongbi Decoction in CIA mouse model.Forty SPF male DBA/1 mice,aged 7-8 weeks,were randomly divided into normal(Normal)group,CIA model(CIA)group,Methotrexate(MTX)group,and Yishen Tongbi Decoction.High-dose(YSTB-H)group and low-dose Yishen Tongbi Decoction(YSTB-L)group,8 in each group.Except for the normal group,mice in other groups need to be immunized twice for the primary and booster immunizations.Mice in each group were treated with drug intervention on the second day after booster immunization(day 21).The mice in the Normal group and the CIA model group were given 100 μl of 0.9%normal saline by gavage every day.The mice in the YSTB-H group and the YSTB-L group were gavaged with 20.6 g/kg/d and 10.3 g/kg/d of Yishen Tongbi Decoction solution,respectively.The mice in the MTX group were gavaged with methotrexate solution 1 mg/kg/q3w.Mice in the YSTB-H,YSTB-L and MTX groups were administered 100 μl by gavage each time according to the above concentrations,and the drugs were continuously intervened for 30 days.During the medication intervention,the general condition,body weight,arthritis index(AI)score,and swollen foot thickness of the mice were observed and recorded every 3 days.After administration,the levels of IL-6,IL-1β,TNF-α,IL-17,TGF-β,and IL-10 inflammatory factors in the serum of mice were detected by ELISA;thymus and spleen indexes were calculated;Knee and ankle joints of mice and bone microstructure scoring;HE staining to observe the structure of mouse knee and ankle joints and pathological scoring;Safranin-fast green staining to observe the cartilage erosion of mouse ankle joints;Immunohistochemistry The positive expression of STAT3 and P-STAT3 in mouse ankle joints was detected by the method.2.Cell experiment:To explore the anti-inflammatory regulatory effect and mechanism of Yishen Tongbi Decoction on RAW264.7 cells.The LPS-induced RAW264.7 cell inflammation model was constructed,the effect of Yishen Tongbi Decoction on the viability of RAW264.7 cells was detected by CCK8 method,and the safe dose of Yishen Tongbi Decoction was screened;the cells were detected by fluorescence microscope and multi-function microplate reader.The fluorescence intensity of reactive oxygen species(ROS)in the cells;the expression level of nitric oxide(NO)in cells was detected by Griess method;the secretion levels of IL-6 and TNF-α in cell supernatants were detected by EILSA method;RT-PCR technology to detect the gene expression levels of IL-6,IL-1 β,TNF-α and iNOS in cells;Western Blot to detect iNOS,COX-2,P-JAK1/JAK1,P-JAK2/JAK2,P-JAK3 Expression levels of/JAK3,P-STAT3/STAT3,SOCS1 and SOCS3.Results1.Animal experiments(1)General situationThe mice in the Normal group were in good mental state,eating normally,with bright coat color,no swelling of the feet,and good mobility of the limbs.Mice in the CIA model group had poor mental state,reduced food intake,irritable state,dull coat color,obvious swelling of ankle joints,decreased mobility of limbs,and a few had erythema and ulceration on the back of the feet.Compared with the mice in the CIA model group,the mental state,food intake,coat color gloss,swelling degree of ankle joints,and limb mobility of the mice in the YSTB-H group,the YSTB-L group and the MTX group were improved to varying degrees.(2)Body weight,swollen foot thickness,Arthritis Index(AI)scoreCompared with the mice in the Normal group,the thickness of the swollen foot in the CIA model group increased,the AI score increased,and the body weight decreased after the booster immunization,and the differences were statistically significant from the 24th day,24th day,and 27th day respectively(P<0.001,P<0.001,P<0.001).Compared with the mice in the CIA model group,in terms of body weight,the YSTB-H group,the MTX group and the YSTB-L group had different degrees of weight gain from the 36th day,the 39th day and the 42nd day,respectively(P<0.05,P<0.01 P<0.01);in terms of the thickness of the foot swelling,the thickness of the foot swelling in the CIA model group reached the peak on the 39th day,and the YSTB-H group,the MTX group and the YSTB-L group started from the 27th day respectively.,the 33rd day,and the 36th day,the thickness of the swollen foot decreased and there was a statistical difference(P<0.05,P<0.01,P<0.01).From 39 days,the AI score decreased in different degrees,and the difference was statistically significant(P<0.05,P<0.05).(3)Levels of inflammatory factorsCompared with the CIA model group,the levels of TNF-α,IL-6,IL-17 and IL-1βdecreased in different degrees after intervention in the YSTB-H group,YSTB-L group and MTX group,while the levels of TGF-β and IL-1β decreased in different degrees.The level of-10 increased in different degrees,and the difference was statistically significant(P<0.05).Compared with the MTX group,the YSTB-H group was more effective in reducing the pro-inflammatory factors IL-6 and IL-17 and increasing the level of the anti-inflammatory factor TGF-β(P<0.05).(4)Thymus and spleen indexCompared with the mice in the CIA model group,the thymus and spleen indices in the YSTB-H group,the YSTB-L group and the MTX group decreased to varying degrees after intervention,and the differences were statistically significant(P<0.05).Compared with MTX group,the thymus and spleen index of YSTB-H group and YSTB-L group had no statistical difference(P>0.05).(5)Safranin-fast green stainingThe structure of the ankle joint of the mice in the Normal group was complete,and the cartilage matrix was clearly stained with safranine,which was clearly distinguished from the osteogenic tissue.In the CIA model group,the ankle joint structure was disordered,the thickness of the cartilage was thinned,the integrity of the bone was destroyed,the synovium invaded the cartilage site,the cartilage matrix was lightly stained or even depigmented with safranine,and the red coloring was not obvious.Compared with the mice in the CIA model group,the ankle joint structure of the YSTBH group,the YSTB-L group and the MTX group after intervention was relatively complete,and the cartilage matrix showed different degrees of red staining.(6)Hematoxylin-eosin(HE)staining and scoringIn the Normal group,the ankle joint surface and knee joint surface were round and complete,the cartilage thickness was moderate,the joint cavity space was large,the bone structure was clear,and there was no obvious synovial cell proliferation,no inflammatory cell infiltration and other pathological changes.In the CIA model group,the ankle joint surface and knee joint surface are uneven,the cartilage thickness is thin and the erosion is serious,the joint space is narrowed,the bone destruction is obvious,the synovial cells proliferate,and the arrangement is loose and disordered,and the inflammatory cell infiltration is obvious.Compared with the mice in the CIA model group,the joint space of the ankle joint surface and the knee joint surface after intervention in the YSTB-H group,the YSTB-L group and the MTX group showed different degrees of widening,the cartilage thickness increased and the degree of erosion was improved.Quality damage was reduced,synovial cell proliferation and inflammatory cell infiltration were reduced.Compared with the mice in the CIA model group,the scores of inflammatory infiltration,synovial hyperplasia,cartilage erosion and bone destruction in the YSTB-H group,YSTB-L group and MTX group were decreased to varying degrees after intervention,and the differences were statistically significant(P<0.05).(7)micro-CT scan and bone microstructure parametersThe bone cortical surface of the knee joint and ankle joint of the mice in the Normal group was clear and smooth,the joint space was normal,the bone density was similar to the surrounding bone,and the articular bone structure was complete and easy to identify.In the CIA model group,the surface of the bone cortex of the knee joint and ankle joint of the mice was sparse and rough,the joint space increased,there were a large number of honeycomb-like destruction cavities,the bone density was significantly reduced,and the joint bone structure was disordered and irregular.Compared with the mice in the CIA model group,the knee and ankle cortical surfaces of the mice in the YSTB-H group,the YSTB-L group and the MTX group were relatively intact,the honeycomb-like destruction cavities were reduced,and the degree of osteolytic loss was reduced,the articular bone structure is relatively regular and complete.Compared with mice in CIA model group,the bone mineral density(BMD),bone volume fraction(BV/TV),trabecular bone thickness(Tb.Th)and trabecular bone thickness(Tb.Th)in YSTB-H group,YSTB-L group and MTX group after intervention The number(Tb.N)increased in different degrees,while the trabecular bone separation(Tb.sp)decreased,and the differences were statistically significant(P<0.05).Compared with the MTX intervention group,the YSTB-H dose group was more effective in improving BMD and BV/TV(P<0.05).(8)ImmunohistochemistryThere was no obvious brown-yellow staining of STAT3 and P-STAT3 in the ankle joints of mice in the Normal group.The joint structure of the mice in the CIA model group was severely damaged,and there were a large number of STAT3 and P-STAT3 positive staining in the nucleus and the cytoplasm of the synovial tissue,showing yellow or brownish yellow and darker staining.Compared with the CIA model group,the positive expression sites of STAT3 and P-STAT3 in the ankle joints of the mice in the YSTB-H group,the YSTB-L group and the MTX group decreased,and the degree of staining decreased.2.Cell Experiments(1)CCK8 resultCompared with the blank(Control)group,the cell viability of YSTB showed a slight upward trend at the dose of 12.5-50 μg/mL,and had no significant effect on the viability of RAW264.7 cells in the concentration range of 400μg/mL,and RAW264.7 at the concentration of 600-1000μg/mL.Cell viability decreased(P<0.05).Therefore,we selected three concentration gradients of 100 μg/mL,200μg/mL and 400 μg/mL as the low,medium and high dose groups of Yishen Tongbi Decoction in the RAW264.7 cell experiment.(2)ROS resultsThe green fluorescence intensity of RAW264.7 cells induced by LPS(100ng/ml)increased significan tly,and the fluorescence intensity of ROS increased.Compared with cells in LPS model group,YSTB(400 μg/mL)group and YSTB(200μg/mL)group could significantly reduce the fluorescence intensity of RAW264.7 cells and inhibit the expression level of ROS(P<0.05);YSTB(100 μg/mL)The group had no obvious inhibitory effect on ROS secretion in RAW264.7 cells(P>0.05).(3)NO resultCompared with the LPS model group,the content of NO secreted by RAW264.7 cells in the YSTB(400 μg/mL)group and the YSTB(200 μg/mL)group decreased to varying degrees after intervention,and the differences were statistically significant(P<0.05);YSTB(100μg/mL)group had no obvious inhibitory effect on NO(P>0.05).(4)ELISA resultsCompared with the LPS model group,the expression levels of TNF-α and IL-6 in RAW264.7 cells in the YSTB(400μg/mL)group and the YSTB(200 μg/mL)group decreased to varying degrees after intervention,and the differences were statistically significant(P<0.05);YSTB(100μg/mL)group had no significant effect on reducing the expression levels of TNF-α and IL-6(P>0.05).(5)RT-PCR resultsCompared with the LPS model group,the levels of TNF-α,IL-6,IL-1 β,and iNOS genes in the YSTB(400 μg/mL)and YSTB(200 μg/mL)groups decreased to varying degrees,and the differences were statistically significant(P<0.05);YSTB(100 μg/mL)group had no significant effect on the expression levels of TNF-α,IL-6,IL-1 β and iNOS genes after intervention(P>0.05).(6)Western-blot resultsCompared with the control group,the expression of iNOS,COX-2,P-JAK1/JAK1,PJAK2/JAK2,P-JAK3/JAK3 and P-STAT3/STAT3 in RAW264.7 cells was significantly increased after LPS(100ng/ml)induction.increased,the expression level of SOCS3 decreased(P<0.05),but had no significant effect on the expression of SOCS1(P>0.05).Compared with the LPS model group,YSTB inhibited the protein expression of iNOS,COX-2,P-JAK2/JAK2,P-JAK3/JAK3 and P-STAT3/STAT3 to different extents,and activated the protein expression of SOCS3(P<0.05),but had a negative effect on the expression of SOCS3.The inhibitory effect of P-JAK1/JAK1 was not obvious(P>0.05).Conclusion1.Yishen Tongbi Decoction can improve the symptoms of arthritis in CIA mice,and its mechanism may be by regulating the serum levels of TNF-α,IL-6,IL-17,IL-1 β,TGFβ and IL-10 The secretion of inflammatory factors inhibits the expression of STAT3 and phosphorylation levels,thereby reducing joint inflammation and inhibiting bone destruction.2.Yishen Tongbi Decoction can reduce the secretion levels of inflammatory mediators NO,iNOS,COX-2,ROS and inflammatory factors IL-6,TNF-α,IL-1 β in the RAW264.7 cell inflammation model,which may be related to the significant inhibition.The phosphorylation of JAK2,JAK3,and STAT3 proteins is related to the up-regulation of the negative feedback factor SOCS3 protein expression.3.Yishen Tongbi Decoction may exert anti-inflammatory and anti-bone destruction effects by regulating the JAK/STAT3/SOCS pathway,so as to achieve the purpose of treating RA. |
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