Objective Cheng's Juanbi Decoction(CSJBD)is a classic traditional Chinese medicine formula for treating rheumatoid arthritis(RA),exhibiting significant clinical efficacy,but the underlying mechanisms remain unclear.We investigated whether CSJBD inhibited RA pathology by blocking the WTAP-Wnt7b-Wnt/β-catenin signaling axis using a collagen-induced arthritis(CIA)mouse model and fibroblast-like synoviocytes(FLSs)derived from RA patients(RA FLSs)and examined the underlying mechanisms.Methods We conducted in vivo experiments.Male C57BL/6 mice weighing 17 to 20 g were used to establish the CIA model.The mice were assigned to 6 groups,including the normal group,the model(CIA)group,the model+CSJBD-L(8.1 g/kg)group,the model+CSJBD-M(16.2 g/kg)group,the model+CSJBD-H(32.4 g/kg)group,and the model+leflunomide(LEF)(0.05 mg/10 g)group,with 10 mice in each group.CSJBD was administered twice daily via gastric gavage,while LEF was administered once daily via gastric gavage,for a duration of 28 days.We also conducted in vitro experiments.RA FLSs were assigned to 4 groups,including the RA FLSs+CSJBDS-L group receiving 10%CSJBDS-containing serum,the RA FLSs+CSJBDS-M group receiving 15%CSJBDS-containing serum,the RA FLSs+CSJBDS-H group receiving 20%CSJBDS-containing serum,and the RA FLSs+NC group(negative control).To study whether WTAP regulated Wnt7b,RA FLSs were divided into the RA FLSs group,the RA FLSs+si-WTAP#3 group,the RA FLSs+si-WTAP#3+Wnt7b-OE group,and the RA FLSs+si-WTAP#3+Wnt7b-NC group.To study the underlying mechanism by which CSJBT affected RA FLSs,RA FLSs were divided into the RA FLSs group,the RA FLSs+CSJBDS-M group,the RA FLSs+CSJBDS-M+Wnt7b-OE group,and the RA FLSs+CSJBDS-M+NC group.We used ultra-high performance liquid chromatography(UPLC)to identify and quantify key monomer compounds from CSJBD as quality criteria for CSJBD preparation.Bioinformatics,CCK-8,RT-qPCR,Western blot,immunofluorescence,and related methods were employed to assess the therapeutic efficacy and underlying mechanisms of CSJBD in treating RA.Results According to the UPLC analysis,ferulic acid,osthole,mulberroside A,notopterol,and gentiopicroside were identified as quality control standards for the preparation of CSJBD formula.CSJBD improved RA pathology in CIA mice,reduced the levels of interleukin(IL)-6,IL-1β,IL-8,and tumor necrosis factor-α(TNF-α)in their serum,and decreased the expression of RA pathological genes MMP3 and fibronectin,with the difference between groups being statistically significant.Bioinformatics analysis suggested that CSJBD might inhibit RA pathology by suppressing the Wnt/β-catenin signaling pathway through Wnt7b.Experimental results showed that the expression of WTAP and Wnt7b was significantly increased in RA.After knocking down WTAP,the expression of Wnt7b was significantly reduced,and the Wnt/β-catenin signaling pathway was also inhibited,with the difference between groups being statistically significant(P<0.05),confirming that WTAP regulated the pathway via Wnt7b.According to experimental verification,CSJBD significantly inhibited the Wnt/β-catenin signaling pathway and the proliferation of RA FLSs.Wnt7b overexpression reversed the inhibitory effect of CSJBD on the Wnt/β-catenin signaling pathway and the proliferation of RA FLSs,indicating that Wnt7b is the direct target of CSJBD.Conclusion CSJBD inhibits RA pathology by blocking the WTAP-Wnt7b-Wnt/β-catenin signaling axis,with Wnt7b identified as a direct therapeutic target of CSJBD.

AIM:This study will clarify whether Wnt5a can be used as a potential diagnostic and therapeutic target for rheumatoid arthritis(RA)and how Xinfeng capsule(XFC)can improve RA through the Wnt5a/β-catenin signaling pathway.METH-ODS:ELISA and RT-qPCR were used to detect in-flammatory factors and pathological genes in the rat model of AA in vivo to investigate the effect of XFC on AA rats.RT-qPCR was used to verify the core genes and key pathways of XFC regulation pre-dicted by network pharmacology.The regulatory mechanism of XFC on Wnt/β-catenin pathway was elucidated by RT-qPCR.Western blot and immuno-fluorescence in primary AA fibroblast-like synovial cells(FLS)in vitro.RESULTS:XFC significantly de-creased the arthritis score and paw swelling in AA rats,and inhibited joint inflammation in AA rats.XFC decreased the levels of inflammatory factors TNF-α and IL-1 in peripheral blood of AA rats,and inhibited the levels of pathological genes MMP3 and fibronectin in joint synovium and AA FLS of AA rats.Network pharmacology predicts that the Wnt pathway is highly correlated with XFC treatment of RA.At the cellular level,serum containing XFC in-hibited the expression of Wnt pathway-related genes β-catenin,CCND1 and c-Myc.The molecular docking results showed that the key components of XFC had strong binding ability to Wnt5a,and the overexpression of Wnt5a(Wnt5a-ove)in AA FLS in-terfered with the action of XFC.CONCLUSION:The expression of Wnt5a is significantly increased in AA FLS and RA FLS,and XFC can inhibit the activation of Wnt/β-catenin signaling pathway to improve RA by binding with Wn5a,providing a new therapeutic mechanism for XFC to improve RA.

Objective Based on the secreted frizzled-related protein 2(SFRP2)-Wnt/β-catenin signaling pathway,this study explored the effect and mechanism of Cuiru Keli(CRKL)in the treatment of postpartum hypogalactia.Methods A rat model of postpartum hypogalactia was established by gavaging 2 mL of 1.6 mg/mL bromocriptine mesylate to female rats on the third day after delivery.Female rats with a delivery time difference of less than 48 hours were selected and randomly assigned to 7 groups,including a normal group(without any modeling or medication),a model group,a CRKL low-dose group of model group model rats receiving CRKL at the dose of 3 g/kg,a CRKL medium-dose group of model rats receiving CRKL at the dose of 6 g/kg,a CRKL high-dose group of model rats receiving CRKL at the dose of 9 g/kg,a positive drug group of model rats receiving domperidone at the dose of 3 mg/kg,and a negative control(NC)group of model rats receiving normal saline.Each group contained 6 rats.Except for the normal and model groups,the remaining 5 groups were continuously administered with the respective intervention drugs at the specified doses by gavage once a day for 10 days.Changes in the total litter mass of the offspring in the 7 groups within 10 days were measured,and HE staining was performed to identify pathological changes in the mammary tissue(MT).Six groups of rats(excluding the positive control group)were used to observe the pathological changes of eosinophils in pituitary tissue.ELISA was performed to determine the content of prolactin(PRL)in serum,immunohistochemical staining was used to determine the expression of prolactin receptor(PRLR)in MT,and RT-qPCR was used to determine the mRNA expression of genes related to lactation in MT.Network pharmacology and molecular docking were used to study the therapeutic effect and mechanism of CRKL on postpartum hypogalactia,particularly whether it acted through the SFRP2-Wnt/β-catenin signaling pathway.The mechanism of CRKL treatment was further validated by detecting mRNA(RT-qPCR)and protein expression(Western blot)of related pathway genes.Cell experiments were conducted using primary culture rat mammary epithelial cells(RMEC)from rat MT.RMEC were divided into four groups,including a normal group(primary culture RMEC,untreated),SFRP2 overexpression group(primary cultured RMEC treated with SFRP2 overexpression vector),SFRP2 overexpression+CRKL group(receiving treatment for SFRP2 overexpression group plus 10% drug-containing serum),and negative control group(primary culture RMEC treated with empty vector).The effect of CRKL on the expression of lactation-related genes FASN,CSN2,and GLUT1 mRNA after SFRP2 overexpression was detected by RT-qPCR.Results In this study,CRKL was administered at a dose of 3 g/kg in the CRKL low-dose group,6 g/kg in the medium-dose group,and 9 g/kg in the high-dose group(P<0.05 or P<0.01).Compared with the model group,CRKL at all doses significantly increased the total litter weight gain of the offsprings within 10 days(P<0.05 or P<0.01),and effectively increased lactation(P<0.01),the area of mammary lobules,and the size and filling of acinar cavities.CRKL at all doses also increased the number of eosinophils that secreted PRL in the pituitary gland of the postpartum hypogalactia rat model,and increased the content of PRL in the serum(P<0.05 or P<0.01).CRKL promoted the secretion and expression of PRL in postpartum hypogalactic model rats.In addition,it significantly promoted the expression of genes related to milk fat,milk protein,and lactose synthesis in MT(P<0.05 or P<0.01).Network pharmacology predicted that the Wnt signaling pathway might be a key pathway for CRKL in treating postpartum hypogalactia.The molecular docking results showed that related chemical components in CRKL had good binding ability with CCND1 and SFRP2.Compared with the model group,CRKL at all doses inhibited the expression of SFRP2 gene in vivo(P<0.01)and activated the mRNA and protein expression of CCND1 and c-Myc in the Wnt/β-catenin signaling pathway in MT(P<0.05 or P<0.01).Cell experiments showed that,compared to the normal group,SFRP2 overexpression reduced the mRNA expression of milk synthesis-related genes FASN,CSN2,and GLUT1 in RMEC(P<0.01).The CCK8 results indicated that 10% of the drug-containing serum was the effective concentration administered to cells(P<0.01).After administering drug-containing serum,the expression of the lactation-related genes FASN,CSN2,and GLUT1 were up-regulated(compared with the SFRP2 overexpression group,P<0.01).Conclusion CRKL alleviates postpartum hypogalactia through the SFRP2-Wnt/β-catenin signaling pathway.SFRP2 might be a potential new target for the diagnosis and treatment of postpartum hypogalactia.This reveals a new mechanism of CRKL in treating postpartum hypogalactia and promotes its clinical application.

AIM: To investigate the effect of an effective component total sterone (TSR) of Echinops latifolius Tausch, the main component of a Chinese patent medicine Cuiru Keli (national drug standard WS3-413 (Z-085)-2003 (Z), on lactation and its possible mechanism. METHODS: After mating between male and female SD rats, 60 female rats were randomly divided into normal control group, model group, TSR low-dose and high-dose groups and prolactin granule positive control group, with 12 female rats in each group and 8 newborn rats in each nest. In addition to the normal control group, the rats in each group were intraperitoneally injected with levodopa 2 mg/kg once a day for 7 days from the second day of delivery. The rats in the normal control group were given normal saline by gavage once a day for 14 days. From the beginning of self-sufficiency, the single lactation of the female rats was measured every day until the 14th day, and then the female rats in each group were killed. Pathological HE staining was used to observe the morphological changes of mammary gland tissue in each group. ELISA was used to detect the levels of serum prolactin (PRL) and 5-hydroxytryptamine (5-HT). Immunohistochemistry was used to detect the distribution of PRL in mammary gland tissue of each group. Furthermore, Real-time qPCR was used to detect the expression of milk protein, milk fat related genes β-casein, FAS, ACC and the expression of canonical Wnt signaling pathway related genes β-catenin, c-Myc, CCND1, SFRP4, DNMT1, MeCP2 in mammary gland of each group. RESULTS: Both low and high dose TSR could significantly increase the single lactation volume, improve the pathological morphology of mammary gland, and increase the serum levels of PRL and 5-HT. TSR increased the distribution of PRL and up-regulated the expression of milk protein, milk fat related genes β-casein, FAS, ACC and canonical Wnt signaling pathway related genes β-catenin, c-Myc, CCND1, SFRP4, DNMT1, MeCP2.CONCLUSION: TSR can significantly promote lactation in lactation deficient rats, and its mechanism may be related to promoting the release of PRL and 5-HT in serum, increasing the distribution of PRL in mammary gland, up-regulating the milk protein and milk fat related genes and activating the canonical Wnt signal.

ABSTRACT OBJECTIVE：To study the improvement effect of Periplaneta americana extract Ento-A on damp-heat ulcerative colitis（UC）model rats. METHODS：Totally 70 rats were randomly divided into normal control group（n＝8）and modeling group （n＝62）. The damp-heat UC model was induced in modeling group by high sugar，high fat，spicy diet combined with 2，4， 6-trinitrobenzene sulfonic acid enema. 48 modelrats were randomly divided into model control group，mesalazine group （300 mg/kg），Changyanning group（300 mg/kg）and Ento-A low-dose，medium-dose and high-dose（50，100，200 mg/kg，calculated by the extract），with 8 rats in each group. Normal control group and model control group were given normal saline intrsgastrically，and other groups were given relevant medicine intragastrically，once a day，for consecutive 14 days. After last administration， disease activity index （DAI）， colonic mucosal injury index （CMDI） and histopathological score （HS）of rats were determined. The spleen index，liver index and colon index in rats were determined. The serum levels of IL-8，IL-17，SOD and MDA，colonic levels of IL-2，PGE2 and MPO were detected by ELISA. RESULTS：Compared with normal control group，the DAI score，CMDI score，HS score，colonic index，the serum levels of IL-8，IL-17 and MDA， colonic levels of MPO and PGE2 were increased significantly（P＜0.01）；serum level of SOD and colonic level of IL-2 were decreased significantly （P＜0.01）. Compared with model control group，DAI score，CMDI score，serum levels of IL-17 and MDA，colonic levels of PGE2 were decreased significantly in Ento-A high-dose groups（P＜0.05 or P＜0.01），while serum level of SOD and colonic level of IL-2 were increased significantly（P＜0.01）. CMDI score and HS score，serum levels of IL-8，IL-17 and MDA，colonic levels of PGE2 and MPO were decreased significantly in Ento-A medium-dose group（P＜0.05 or P＜0.01）， while colonic level of IL-2 was increased significantly（P＜0.01）. HS score，serum levels of IL-17 and MDA，colonic levels of MPO and PGE2 were decreased significantly in Ento-A low-dose group（P＜0.05 or P＜0.01），while serum level of IL-2 was increased significantly（P＜0.01）. CONCLUSIONS：P. americana extract Ento-A may play improvement effect on damp-heat UC rats by regulating immune system balance and reducing inflammatory damage.

OBJECTIVE： To study the improvement effects of Bee venom（BV） plastics on experimental cerebral thrombosis in rats. METHODS： Totally 96 SD rats were randomly divided into sham operation group （normal saline）， model group （plastics blank matrix）， Nimodipine group （positive drug， 4.00 mg/kg） and BV plastics low-dose， medium-dose， high-dose groups （1.67， 3.33， 6.67 mg/kg）， with 16 rats in each group. Rats in sham operation group and Nimodipine group were given medicine intragastrically， while rats in model group and BV plastics groups were given medicine by transdermal smearing. After 5 days of continuous administration， the experimental cerebral thrombosis model was established by ligating the right external carotid artery and pterygomandibular artery， and injecting compound thrombus inducer into the internal carotid artery. The wet mass ratio of right brain to left brain was measured to investigate the degree of brain edema on the infarcted side. The content of Evans blue （EB） in the left and right hemispheres of rats was determined by ultraviolet spectrophotometry to investigate the cerebral vascular permeability. Blood rheology and coagulation function indicators of rats were measured. The pathological changes of brain tissue in rats were observed by HE staining， and the number of survival neuron cells was counted. RESULTS： Compared with the indexes of sham operation group， the cerebral thrombosis model was established successfully. Compared with model group， the area of blue staining in the right brain （infarcted side） of rats in BV plastics groups was significantly reduced， and the right brain/left brain wet mass ratio and the content of EB in the right brain tissue were significantly reduced （P＜0.05 or P＜0.01）. The whole blood viscosity and Casson viscosity of rats in BV plastics groups， and the plasma viscosity of rats in BV plastics medium-dose and high-dose groups decreased significantly （P＜0.01）. PT and APTT of rats were prolonged significantly in BV plastics medium-dose group （P＜0.01）. The pathological changes of brain tissue in rats in BV plastics groups were significantly alleviated. The arrangement of neuron cells was more orderly， the shape and structure of cells were clear， the nucleolus was clear， the membrane was intact， and the number of survival neuron cells was significantly increased （P＜0.01）. CONCLUSIONS： BV plastics can alleviate brain edema， inhibit cerebral vascular permeability， improve hemorheology and coagulation function indicators of rats after the formation of cerebral thrombosis， and alleviate nerve cell injury after ischemia.

OBJECTIVE: To explore the eff ect of pulchinenoside (PULC) on fi broblast-like synoviocytes (FLS) apoptosis in adjuvant arthritis (AA) rats. METHODS: A total of 60 SD rats were randomly divided into 8 groups: A normal control group, an AA group, a low PULC group (50 mg/kg), a middle PULC group (100 mg/kg) or a high PULC group (150 mg/kg) and an ibuprofen (8 mg/kg) group (n=10 per group). FLS from the AA rats was cultured. The expression of Bcl-2, Bax, caspase-3 and the FLS proliferation were detected by the real time qPCR and MTT, respectively. The expression of IL-6 and IL-8 in culture medium was detected by ELISA. RESULTS: Compared with the AA group, the Bcl-2 expression was down-regulated (all P<0.05), the Bax and caspase-3 expression was up-regulated (all P<0.05), and the FLS proliferation was inhibited (all P<0.05). The IL-6 and IL-8 expression was suppressed in the FLS in the PULC groups at different dosages (all P<0.05) as well as in the ibuprofen group (P<0.05). CONCLUSION PULC may inhibit the FLS proliferation in AA rats by increase in FLS apoptosis.
Animals ; Apoptosis ; drug effects ; Arthritis, Experimental ; Caspase 3 ; metabolism ; Fibroblasts ; cytology ; drug effects ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pulsatilla ; chemistry ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; cytology ; bcl-2-Associated X Protein ; metabolism

Objective:To determine the effect of Chuju total flavonoids (CJTF) on the secreted frizzled-related protein 4 (SFRP4) expression in Wnt pathway in rheumatoid arthritis (AR) model rats. Methods:hTe role of CJTF in the treatment of AR model rats was evaluated by rat arthritis score and paw edema score. The expression regulation of the SFRP4,β-catenin and C-myc in Wnt pathway in AR model rats was detected by RT-PCR and Western blot atfer CJTF gavage treatment. Results:Atfer CJTF treatment, the rat arthritis score and paw edema score in AR model rats were signiifcantly decreased when the AR model rats were treated with CJTF, the SFRP4 expression was signiifcantly up-regulated, while theβ-catenin and C-myc gene expression were signiifcantly down-regulated in AR model rat synovial tissues. Conclusion:CJTF has significant therapeutic effect and inhibitory effect on Wnt pathway activation by targeting SFRP4 in AR model rat synovium.