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.

Objective:To investigate the related factors that affect the timing and prognosis of early tracheostomy in patients with multiple rib fractures.Methods:A retrospective case series study was conducted on medical data of 222 patients with multiple rib fractures who underwent tracheostomy in Affiliated Hospital of Yangzhou University from February 2013 to October 2019，including 160 males and 66 females，with the age of 18 to 85 years ［（49.5 ± 16.3）years］. According to the practice management guidelines for tracheostomy timing and the use of propensity score matching technology，there were 118 patients with tracheostomy within 7 days of tracheal intubation （early group） and 104 patients with tracheostomy after 7 days of tracheal intubation （late group） before matching，and there were 87 patients in early group and 87 patients in late group after matching. Data were compared between groups including the gender，age，underlying disease，injury severity score （ISS），Glasgow coma score （GCS），number of fractured ribs，total number of rib fractures （NTRF），first rib fracture，flail chest，traumatic brain injury，combined injuries （spine，maxillofacial，sternum），acute respiratory distress syndrome （ARDS），volume fraction of pulmonary contusion（VPC），blood lactic acid （within 24 hours of admission），hemothorax，pneumothorax，mechanical ventilation time，duration of tracheostomy，time from tracheal intubation to incision，length of hospital stay，length of stay in ICU，closed thoracic drainage，number of fiberoptic bronchoscopy，multi-drug resistant bacteria infection，ventilator-associated pneumonia，antibiotic use time，duration of sedative and analgesic drugs used and 28-day mortality. The multivariate Logistic regression analysis was used to predict independent risk factors for early tracheostomy. The Pearson method was used to compare the relationship between multiple factors. The receiver operating characteristic （ROC） curve was used to predict indicators that affect the prognosis of patients with early tracheostomy，and calculate the best cut-off value. The Kaplan-Meier single factor and COX multivariate survival were used to analyze the relevant factors affecting the 28-day mortality of patients.Results:（1） In early group，the NTRF，ARDS and VPC were higher than those in late group，and the time from tracheal intubation to incision and 28-day mortality rate were lower than those in late group （ P < 0.05），while the two groups showed no significant differences in the gender，age，underlying diseases and ISS （ P > 0.05）. （2） The multivariate Logistic regression analysis showed that there was statistical significance in NTRF （ OR = 1.775，95% CI 1.439-2.188），ARDS（ OR = 3.740，95% CI 1.441-9.711），VPC （ OR = 1.087，95% CI 1.052-1.124） （ P < 0.05）； the Pearson method analysis showed a significant correlation between VPC and NTRF （ r = 0.369， P < 0.05） and a low degree of correlation between ARDS and VPC （ r = 0.179， P < 0.05），but there was no significant correlation between ARDS and NTRF （ r = 0.132， P > 0.05）. （3） The ROC curve analysis showed that the area under the curve （AUC） of the VPC and NTRF ［AUC = 0.832 （95% CI 0.770-0.893），AUC = 0.804 （95% CI 0.740-0.868）］ were significantly higher than those of the number of rib fractures ［AUC = 0.437（95% CI 0.352-0.523），GCS ［AUC = 0.519 （95% CI 0.432-0.605）］ and ISS ［AUC = 0.484 （95% CI 0.398-0.571）］ （ P < 0.05）. After calculating the Yorden index，the best cut-off value for VPC was 23.9，and the best cut-off value for NTRF was 8.5. （4） The Kaplan-Meier single factor and multivariate COX model survival analysis showed that the 28-day survival ratio of patients with early tracheostomy was significantly better than that of late tracheostomy （ P < 0.05）. Conclusions:The NTRF，ADRS and VPC are independent risk factors for the timing and prognosis of early tracheostomy. There is a significant correlation between VPC and NTRF. The VPC ≥ 23.9% and or NTRF ≥ 8.5 can be used to predict early tracheostomy in patients with multiple rib fractures. Early tracheostomy may benefit the 28-day survival of patients with multiple rib fractures.

Objective To investigate the protective effect and its mechanism of infradiaphragmatic stasis-expelling decoction on hepatic fibrosis in rats. Method One hundred and ten SD rats were divided into four groups:the normal control group ,model group ,low dose group and high dose group of infradiaphragmatic stasis-expelling decoction. In addition to the normal control group,rats in other groups were subcutaneously given pure CCl4 to set up the liver fibrosis model. Twelve weeks later,the serum liver biochemical indexes,including ALT,AST, ALB,TP and T-bil,the Ishak score about liver fibrosis ,the positive expression of TIMP-1 were compared among groups. Results Compared with the fibrotic group,the levels of serum ALT,AST and T-bil were lower in low dose group,the level of serum TP and ALB were increased in high dose group Levels of serum ALT,AST and T-bil were significantly lowered in high dose group,the levels of serum of TP and ALB were significantly increased in high dose group. Compared with model group,the Ishak score about liver fibrosis was significantly lowered(P<0.05). Numbers of cells with positive expression of TIMP-1 in liver tissue was reduced. Conclusion Infradiaphragmatic stasis-expelling decoction could inhibit the expression and activity of TIMP-1 contributing to the effect of antiliver fibrosis.