ObjectiveTo investigate the effect and underlying mechanism of the Wulao Qisun prescription on pathological new bone formation in ankylosing spondylitis （AS）. MethodsSynovial fibroblasts were isolated from the hip joints of AS patients and observed under a microscope to assess cell morphology. The cells were identified using immunofluorescence staining. The isolated AS fibroblasts were divided into blank group， low drug-containing serum group， medium drug-containing serum group， high drug-containing serum group， and positive drug group. After drug intervention， cell proliferation was measured using the cell counting kit-8 （CCK-8） assay to observe fibroblast growth and determine the optimal intervention time. Alkaline phosphatase （ALP） activity was measured using the alkaline phosphatase assay. Protein expression of osteocalcin （OCN）， osteopontin （OPN）， and runt-related transcription factor 2 （Runx2） was detected by Western blot. The mRNA expression levels of Wnt5a， β-catenin， and Dickkopf-1 （DKK-1） were measured by real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsCompared with the blank group， each drug-containing serum group of Wulao Qisun prescription and the positive drug group inhibited the proliferation of AS fibroblasts and reduced ALP expression （P<0.01）. Compared with the blank group， the low drug-containing serum group of Wulao Qisun prescription downregulated β-catenin mRNA expression （P<0.05）. The medium and high drug-containing serum groups and the positive drug group significantly downregulated Wnt5a and β-catenin mRNA expression （P<0.05， P<0.01）， with the positive drug group showing the most pronounced effect （P<0.01）. The high drug-containing serum group and the positive drug group significantly upregulated DKK-1 mRNA expression （P<0.01）. Compared with the blank group， the low drug-containing serum group of Wulao Qisun prescription inhibited the expression of OPN and Runx2 proteins （P<0.05， P<0.01）， while the medium and high drug-containing serum groups and the positive drug group inhibited the expression of OCN， OPN， and Runx2 proteins （P<0.05， P<0.01）. ConclusionThe Wulao Qisun prescription can inhibit the proliferation and osteogenic differentiation of AS fibroblasts， thereby delaying the formation of pathological new bone in AS. The possible mechanism involves the regulation of Wnt/β-catenin-related gene expression， further inhibiting the transcription of downstream target genes.

Objective To investigate the effect and mechanism of lentivirus-mediated overexpression of suppressors of cytokine sig-naling 3(SOCS3)on the heart of rats with experimental autoimmune myocarditis(EAM).Methods Thirty Lewis rats were randomly di-vided into a control group,EAM group and EAM+SOCS3 group.The EAM rat model was established by subcutaneous injection of cardiac myosin.On day 0 of the experiment,the EAM+SOCS3 group was injected with lentivirus overexpressing SOCS3 via the tail vein,and the EAM group was injected with an equal amount of empty vector lentivirus.On day 21 of the experiment,all rats underwent echocardio-graphy to evaluate cardiac function and were then euthanized.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of myocardial tissue.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of serum cardiac troponin T,creatine kinase-myocardial band,B-type natriuretic peptide,and myocardial interleukin-6,interleukin-1 β and tumor necrosis fac-tor-α.Immunohistochemistry,real-time quantitative PCR(RT-qPCR)and Western blot(WB)were used to detect the expressions of SOCS3,Janus kinase 2(JAK2),signal transducer and activator of transcription 3(STAT3),monocyte chemoattractant protein-1(MCP-1),and transforming growth factor-β(TGF-β)in myocardial tissue.Results The myocardial pathological score,serum lev-els of cardiac troponin T,creatine kinase-myocardial band,B-type natriuretic peptide,and myocardial levels of interleukin-6,inter-leukin-1β,and tumor necrosis factor-α in the EAM group were significantly higher than those in the control group(P＜0.05).The cardiac function of the EAM group was significantly lower than that of the control group(P＜0.05).The mRNA and protein expressions of myocardial JAK2,STAT3,MCP-1 and TGF-β in the EAM group were significantly higher than those in the control group(P＜0.05).These indexes in the EAM+SOCS3 group were significantly restored compared with those in the EAM group(P＜0.05).The mRNA and protein expressions of SOCS3 in the myocardial tissue of the EAM+SOCS3 group were significantly higher than those in the EAM group(P＜0.05).Conclusion Overexpression of SOCS3 may ameliorates the inflammatory response of the myocardium in EAM rats and im-prove cardiac function by inhibiting the JAK2/STAT3 signaling pathway and downregulating the expressions of MCP-1 and TGF-β.

Osteoporosis(OP)is a prevalent metabolic bone disease with a complex pathogenesis that has not yet been fully elucidated.Recent studies have revealed that the Toll-like receptor 4(TLR4)signaling pathway plays a significant role in the development and progression of OP.TLR4,a crucial immune receptor primarily expressed in immune cells,is involved in inflammatory responses and immune regulation.The TLR4 signaling pathway influences bone metabolism and remodeling through multiple mechanisms.Therefore,investi-gating the role of the TLR4 signaling pathway in OP is of great significance for its prevention and treatment.Re-search targeting the TLR4 signaling pathway provides novel insights and approaches for OP therapy.Future studies should further explore the mechanisms of the TLR4 signaling pathway,develop therapeutic agents that modulate this pathway,and validate their efficacy in OP through clinical trials,thereby offering more options for the clinical management of OP.

AIM:To investigate the therapeutic effect of Qingre sanzhuo decoction on rats with hyperuri-caemia combined with gouty arthritis and its effect on TLR4/Myd88/NF-κB signalling pathway.METHODS:Forty-eight SD male rats were randomly divided into blank,model,and colchicine groups(0.3 mg·kg-1·d-1),and Origre sanzhou decotion low,medium and high-dosage groups(7.42,14.85,29.70 g·kg-1·d-1),which were treated with the modified Coderre method for hyperuricemia combined with acute gouty arthritis via gavage of yeast paste combined with potassium oxa-late,which was used for the treatment of acute gouty arthritis combined with hyperuricemia.A composite rat model of acute gouty arthritis was constructed by combining yeast paste with potassium oxalate gavage to cause hyperuricaemia,combined with the modified Coderre method.After 7 days of intervention,the circumference of the right ankle joint of rats was measured and the swelling of the ankle joint was calculated,the blood uric acid(HUA)level of rats was determined by biochemical method,the histopatho-logical and morphological changes of the synovial membrane of the ankle joint of rats were examined by HE staining,and the serum levels of inflammatory factors,tumour necrosis factor-alpha(TNF-α),inter-leukin-6(IL-6),and IL-1β were determined by enzyme-linked immunosorbent assay(ELISA),and Western blotting was performed to determine the levels of inflammatory factors,TNF-α,and IL-1β.The protein expression levels of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(Myd88),and nuclear factor-κB(NF-κB)in the synovial tissues of the ankle joints of the rats were determined by Western blot method,and the mRNA expression of TLR4,Myd88,and NF-κB in the rat was determined by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).RESULTS:Compared with the blank group,rats in the model group showed significantly lower ankle joint swelling(P<0.01),increased levels of HUA,dis-organised synovial tissue structure,large number of inflammatory cells infiltration,and significantly higher serum levels of TNF-α,IL-6,and IL-1β(P<0.01),and the protein and mRNA expression levels of TLR4,Myd88,and NF-κB in the synovial membrane of the ankle joints of the model group were significantly increased(P<0.01).levels were significantly increased(P<0.01);compared with the model group,joint swelling was significantly reduced in the colchicine group,and the medium-and high-dose groups of Qingre sanzhuo decoction(P<0.05);synovial hyperplasia and inflam-matory cell infiltration were improved in the colchicine group and the medium-and high-dose groups of Qingre sanzhuo decoction,and the HUA and the levels of TNF-α,IL-6,and IL-1β were significantly decreased in the dosing groups(P<0.05,P<0.01),and compared with the model group,the medium-and high-dose groups of Qingre sanzhuo decoction could significantly reduce the expression of TLR4,Myd88,and NF-κB protein and mRNA(P<0.01).CONCLUSION:Qingre sanzhuo decoction reduces the release of inflamma-tory factors by inhibiting the TLR4/Myd88/NF-κB pathway,and plays a role in the treatment of hyper-uricaemia combined with gouty arthritis.

Objective To investigate the effect and mechanism of lentivirus-mediated overexpression of suppressors of cytokine sig-naling 3(SOCS3)on the heart of rats with experimental autoimmune myocarditis(EAM).Methods Thirty Lewis rats were randomly di-vided into a control group,EAM group and EAM+SOCS3 group.The EAM rat model was established by subcutaneous injection of cardiac myosin.On day 0 of the experiment,the EAM+SOCS3 group was injected with lentivirus overexpressing SOCS3 via the tail vein,and the EAM group was injected with an equal amount of empty vector lentivirus.On day 21 of the experiment,all rats underwent echocardio-graphy to evaluate cardiac function and were then euthanized.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of myocardial tissue.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of serum cardiac troponin T,creatine kinase-myocardial band,B-type natriuretic peptide,and myocardial interleukin-6,interleukin-1 β and tumor necrosis fac-tor-α.Immunohistochemistry,real-time quantitative PCR(RT-qPCR)and Western blot(WB)were used to detect the expressions of SOCS3,Janus kinase 2(JAK2),signal transducer and activator of transcription 3(STAT3),monocyte chemoattractant protein-1(MCP-1),and transforming growth factor-β(TGF-β)in myocardial tissue.Results The myocardial pathological score,serum lev-els of cardiac troponin T,creatine kinase-myocardial band,B-type natriuretic peptide,and myocardial levels of interleukin-6,inter-leukin-1β,and tumor necrosis factor-α in the EAM group were significantly higher than those in the control group(P＜0.05).The cardiac function of the EAM group was significantly lower than that of the control group(P＜0.05).The mRNA and protein expressions of myocardial JAK2,STAT3,MCP-1 and TGF-β in the EAM group were significantly higher than those in the control group(P＜0.05).These indexes in the EAM+SOCS3 group were significantly restored compared with those in the EAM group(P＜0.05).The mRNA and protein expressions of SOCS3 in the myocardial tissue of the EAM+SOCS3 group were significantly higher than those in the EAM group(P＜0.05).Conclusion Overexpression of SOCS3 may ameliorates the inflammatory response of the myocardium in EAM rats and im-prove cardiac function by inhibiting the JAK2/STAT3 signaling pathway and downregulating the expressions of MCP-1 and TGF-β.

AIM:To investigate the therapeutic effect of Qingre sanzhuo decoction on rats with hyperuri-caemia combined with gouty arthritis and its effect on TLR4/Myd88/NF-κB signalling pathway.METHODS:Forty-eight SD male rats were randomly divided into blank,model,and colchicine groups(0.3 mg·kg-1·d-1),and Origre sanzhou decotion low,medium and high-dosage groups(7.42,14.85,29.70 g·kg-1·d-1),which were treated with the modified Coderre method for hyperuricemia combined with acute gouty arthritis via gavage of yeast paste combined with potassium oxa-late,which was used for the treatment of acute gouty arthritis combined with hyperuricemia.A composite rat model of acute gouty arthritis was constructed by combining yeast paste with potassium oxalate gavage to cause hyperuricaemia,combined with the modified Coderre method.After 7 days of intervention,the circumference of the right ankle joint of rats was measured and the swelling of the ankle joint was calculated,the blood uric acid(HUA)level of rats was determined by biochemical method,the histopatho-logical and morphological changes of the synovial membrane of the ankle joint of rats were examined by HE staining,and the serum levels of inflammatory factors,tumour necrosis factor-alpha(TNF-α),inter-leukin-6(IL-6),and IL-1β were determined by enzyme-linked immunosorbent assay(ELISA),and Western blotting was performed to determine the levels of inflammatory factors,TNF-α,and IL-1β.The protein expression levels of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(Myd88),and nuclear factor-κB(NF-κB)in the synovial tissues of the ankle joints of the rats were determined by Western blot method,and the mRNA expression of TLR4,Myd88,and NF-κB in the rat was determined by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).RESULTS:Compared with the blank group,rats in the model group showed significantly lower ankle joint swelling(P<0.01),increased levels of HUA,dis-organised synovial tissue structure,large number of inflammatory cells infiltration,and significantly higher serum levels of TNF-α,IL-6,and IL-1β(P<0.01),and the protein and mRNA expression levels of TLR4,Myd88,and NF-κB in the synovial membrane of the ankle joints of the model group were significantly increased(P<0.01).levels were significantly increased(P<0.01);compared with the model group,joint swelling was significantly reduced in the colchicine group,and the medium-and high-dose groups of Qingre sanzhuo decoction(P<0.05);synovial hyperplasia and inflam-matory cell infiltration were improved in the colchicine group and the medium-and high-dose groups of Qingre sanzhuo decoction,and the HUA and the levels of TNF-α,IL-6,and IL-1β were significantly decreased in the dosing groups(P<0.05,P<0.01),and compared with the model group,the medium-and high-dose groups of Qingre sanzhuo decoction could significantly reduce the expression of TLR4,Myd88,and NF-κB protein and mRNA(P<0.01).CONCLUSION:Qingre sanzhuo decoction reduces the release of inflamma-tory factors by inhibiting the TLR4/Myd88/NF-κB pathway,and plays a role in the treatment of hyper-uricaemia combined with gouty arthritis.

AIM:To investigate the inhibitory ef-fect of Zushima ointment on inflammation and bone destruction in CIA mice.METHODS:SPF grade male DBA/1 mice were used,6 were random-ly selected as the normal group,and 18 CIA mice that were successfully modelled were randomly di-vided into the model group,the plaster group(1.0 g/kg),and the fuselage group(0.12 g per time)ac-cording to the random number table method,6 mice in each group,and each administered group was given medication according to the body mass,and saline was given to both the normal and model groups.The normal group and the model group were given saline,and breathable adhesive paper was applied once a day for 4 h/session for 4 consec-utive weeks.The arthritis scoring index was used to observe the changes of arthritis symptoms and ar-thritis index scores of mice in each group.Micro-CT was used to observe the damage of hind paw of mice,real-time fluorescence PCR was used to de-tect the mRNA expression of IL-17,IL-1β and TNF-αin ankle joint tissues,and immunohistochemistry was used to detect the expression of OPG and RANKL proteins in ankle joint tissues,and hematox-ylin-eosin(HE)staining was used to observe the pathological changes of synovial tissues after the treatment.The pathological changes of synovial tis-sue were observed after hematoxylin-eosin(HE)staining,and the changes of osteoclasts in ankle joint tissue were observed by anti-tartaric acid phosphatase(TRAP)method.RESULTS:Compared with the normal group,the arthritis index score of the model mice was significantly higher(P＜0.05).Micro-CT showed severe bone erosion in the hind paws of the mice,destruction of the bone surface and reduction of bone volume.The expression of IL-17,IL-1β and TNF-α mRNA(PCR)in the ankle joint tissues was significantly higher(P＜0.05).Im-munohistochemistry showed that the relative ex-pression of OPG protein in the ankle joint tissues was reduced(P＜0.05).Immunohistochemistry showed a decrease in the relative expression of OPG protein(P＜0.01)and an increase in the rela-tive expression of RANKL protein(P＜0.01).HE re-sults showed moderate inflammatory cell infiltra-tion,swelling of synovial cells,massive formation of vascular opacities and synovial hyperplasia;an increase in the number of osteoclasts,roughness of the surface of articular cartilage tissue,severe bone destruction and thinning of the cartilage lay-er.Compared with the model group,the arthritic symptoms of mice in the cream group and the futa-lin group were relieved and the arthritis index score was reduced;the bone density of the mice's hind paws improved,effectively relieving osteopo-rosis;the expression of IL-17,IL-1β and TNF-αmRNA(PCR)in the ankle joint tissue was signifi-cantly reduced(P＜0.05);the immunohistochemical results showed that the relative expression of OPG protein was increased(P＜0.05),the relative expres-sion of RANKL protein decreased(P＜0.01).HE re-sults showed that synovial cell enlargement was significantly improved,mild inflammatory cell infil-tration,synovial hyperplasia was not obvious;the number of broken bone was reduced,articular car-tilage destruction was significantly improved and relieved,and the thickness of cartilage layer was significantly increased.CONCLUSION:Ancestral hemp poultice relieves local symptoms of RA,re-duces the expression of inflammatory factors and attenuates the inflammatory response,possibly by inhibiting osteoclast differentiation and activation through modulation of the OPG/RANKL signalling axis,which further ameliorates the biological ef-fects of articular bone and cartilage destruction.

Ankylosing spondylitis(AS)is a typical spinal arthritis characterised by inflammatory back pain,which seriously affects the health and quality of life of patients.The clinical efficacy of Chinese medicine in the treatment of ankylosing spondylitis is clear,but the mechanism is not clear,and the existing animal models cannot be well applied to the evaluation of Chinese medicine in the treatment of ankylosing spondylitis.Therefore,this paper summarizes the existing animal models based on Chinese and Western medicine clinical diagnosis,disease characteristics,etiology and Chinese medicine evidence,and finds that among the existing animal models,the proteoglycan-induced arthritis mouse model has a higher Chinese and Western medicine clinical fit than the other models,but lacks the corresponding Chinese medicine evidence model evaluation.The other animal models had a higher Western clinical match,but lacked the characteristics of the Traditional Chinee Medicine(TCM)syndrome.As ankylosing spondylitis is a chronic inflammatory autoimmune disease with complex pathogenic factors,the existing animal models cannot better simulate the clinical symptoms.Therefore,the establishment of animal models of ankylosing spondylitis with the characteristics of Chinese and Western clinical evidence is a future research priority for AS TCM.

Osteoporosis (OP) is a prevalent metabolic bone disease with a complex pathogenesis that has not yet been fully elucidated. Recent studies have revealed that the Toll-like receptor 4 (TLR4) signaling pathway plays a significant role in the development and progression of OP. TLR4, a crucial immune receptor primarily expressed in immune cells, is involved in inflammatory responses and immune regulation. The TLR4 signaling pathway influences bone metabolism and remodeling through multiple mechanisms. Therefore, investigating the role of the TLR4 signaling pathway in OP is of great significance for its prevention and treatment. Research targeting the TLR4 signaling pathway provides novel insights and approaches for OP therapy. Future studies should further explore the mechanisms of the TLR4 signaling pathway, develop therapeutic agents that modulate this pathway, and validate their efficacy in OP through clinical trials, thereby offering more options for the clinical management of OP.

Rheumatoid arthritis(RA)is an autoimmune disease with the basic pathological manifestation of synovial inflammation.Symmetric poly-articular pain and swelling are the main symptoms in clinical practice,and even extra-articular manifestations and comorbidities such as interstitial fibrosis and coronary artery disease are triggered,which seriously affect the quality of life of patients.Traditional Chinese medicine(TCM)has achieved good clinical efficacy in the prevention and treatment of RA with the advantages of multi-pathway,multi-target,multi-component,and less toxic side effects,and plays an important role in the treatment of RA.Recently,many studies have demonstrated that Chinese medicine monomers and Chinese herbal compound can control inflammation,reduce angiogenesis,induce apoptosis of synovial fibroblasts,and inhibit their proliferation,invasion and migration by regulating the PI3K/Akt signaling pathway,so as to play a key role in the treatment of RA.For this reason,the article summarizes current knowledge regarding the PI3K/Akt signaling pathway and its role in RA,as well as summarizes the current research progress of TCM in the treatment of RA by regulating the PI3K/Akt signaling pathway.The aim of this review is to provide theoretical bases for the prevention and treatment of RA and the development of new drugs.