BACKGROUND:Traditional methods of urinary tract reconstruction are limited by donor scarcity,high complication rates,and suboptimal functional recovery.Tissue engineering strategies offer new directions in this field.Since the urinary tract is mainly composed of muscle tissue,the key is to find suitable seed cells and efficiently induce them to differentiate into smooth muscle cells.Comparative studies on the efficacy of different smooth muscle cell induction regimens are still lacking. OBJECTIVE:To isolate,culture,and identify human urine-derived stem cells,and to compare the effects of two different induction protocols. METHODS:Human urine-derived stem cells were isolated from urine samples of 11 healthy adult volunteers by multiple centrifugations.Surface markers were identified by flow cytometry.The multi-directional differentiation potential of human urine-derived stem cells was verified through osteogenic and adipogenic differentiation.Differentiation was induced by transforming growth factor-β1 or transforming growth factor-β1 combined with platelet derived growth factor for 14 days.Immunofluorescence staining and western blot assay were employed to compare the expression differences of smooth muscle-specific proteins(α-SMA and SM22). RESULTS AND CONCLUSION:(1)Urine-derived stem cells were successfully isolated from the eight urine samples of healthy people.These cells exhibit a"rice grain"-like morphology and possess a robust proliferative capacity.(2)Urine-derived stem cells exhibited high expression of mesenchymal stem cell surface markers(CD73,CD90,and CD44)and extremely low expression of hematopoietic stem cell surface markers(CD34 and CD45).These cells did not express CD19,CD105,and HLA-DR.(3)After osteogenic and adipogenic differentiation,the formation of calcium nodules and lipid droplets was observed,with positive staining results from Alizarin Red S and Oil Red O staining.(4)After 14 days of smooth muscle induction culture,immunofluorescence staining revealed that the smooth muscle differentiation rate of urine-derived stem cells treated with a combination of transforming growth factor-β1 and platelet derived growth factor was significantly higher compared to those treated with transforming growth factor-β1 alone(P<0.005).(5)After 14 days of smooth muscle induction culture,western blot assay further demonstrated that the expression levels of α-SMA and SM22 in the transforming growth factor-β1/platelet derived growth factor group were significantly elevated compared to those in the transforming growth factor-β1 only group(P<0.005).These findings confirm that urine-derived stem cells can be non-invasively isolated using multiple rounds of centrifugation.Compared with transforming growth factor-β1 alone,the combination of transforming growth factor-β1 and platelet derived growth factor can improve the efficiency of inducing urine-derived stem cells to differentiate into smooth muscle cells.

OBJECTIVES: To explore the mechanism of cinnamic acid (CA) for improving doxorubicin-induced myocardial injury (DIC) in mice. METHODS: Network pharmacology analysis was used to obtain the key targets of CA and DIC. Male C57BL/6J mice were randomized into Sham, DOX, CA (25, 50 and 100 mg/kg)+DOX, and CA+Ferrostatin-1+DOX groups, and their myocardial function and pathology were examined by echocardiography and HE staining. Serum levels of CK-MB, LDH, MDA, IL-6, TNF‑α and myocardial ROS level were detected, and the expression levels of TLR4 and ferroptosis pathway proteins in myocardial tissue were detected by Western blotting. Cultured murine cardiomyocytes (HL-1 cells) with or without transfection with a small interfering RNA targeting TLR4 (si-TLR4) were treated with DOX or Erastin, and the cellular ROS content was measured by DCFH-DA staining; the expression level of GPX4 was detected using immunofluorescence staining. RESULTS: Network pharmacology analysis suggested that CA may improve DIC through TLR4 signaling. DOX treatment caused obvious myocardial injury in mice, which showed significantly increased serum levels of CK-MB, LDH, MDA, IL-6, TNF-α and myocardial ROS level with decreased myocardial levels of SLC7A11 and GPX4 proteins and increased levels of TLR4 and PTGS2 proteins. All these changes in the mouse models were significantly alleviated by treatment with CA, and the mice receiving CA or ferrostatin-1 treatment exhibited increased myocardial expressions of SLC7A11 and GPX4 proteins and lowered expressions of TLR4 and PTGS2 proteins. In cultured HL-1 cells, treatment with DOX and Erastin both obviously increased intracellular ROS level and decreased cellular GPX4 expression level, and these changes were strongly attenuated by TLR4 interference. CONCLUSIONS CA, as a potent herbal monomer, can effectively alleviate DIC in mice by inhibiting TLR4-mediated ferroptosis.
Animals ; Ferroptosis/drug effects* ; Toll-Like Receptor 4/metabolism* ; Myocytes, Cardiac/metabolism* ; Mice, Inbred C57BL ; Mice ; Male ; Doxorubicin/adverse effects* ; Cinnamates/pharmacology* ; Signal Transduction ; Reactive Oxygen Species/metabolism*

The mandibular condyle is a critical growth center in craniofacial bone development, especially during postnatal stages. Postnatal condyle osteogenesis requires precise spatiotemporal coordination of growth factor signaling cascades and hierarchical gene regulatory networks. Plagl1, which encodes a zinc finger transcription factor, is a paternally expressed gene. We demonstrate that PLAGL1 is highly expressed in cranial neural crest cell (CNCC)-derived lineage cells in mouse condyles. Using the CNCC-derived lineage-specific Plagl1 knockout mouse model, we evaluate the function of PLAGL1 during postnatal mouse condyle development. Our findings show that PLAGL1 contributes significantly to osteoblast differentiation, and its deficiency impairs osteogenic lineage differentiation, which consequently disrupts mandibular condyle development. Mechanistically, insulin-like growth factor 2 (IGF2) in complex with IGF-binding proteins (IGFBPs) has been identified as the principal PLAGL1 effector responsible for osteogenic regulation during postnatal condyle morphogenesis. Plagl1 deficiency significantly downregulates the IGF2/IGFBP pathway, leading to disordered glucose metabolism, defective extracellular matrix organization, and impaired ossification. Exogenous IGF2 treatment rescues impaired osteoblast differentiation caused by Plagl1 deficiency. In conclusion, the PLAGL1-IGF2 axis is a critical regulator of osteogenesis during mandibular condyle development.
Animals ; Osteogenesis/genetics* ; Insulin-Like Growth Factor II/metabolism* ; Mice ; Transcription Factors/metabolism* ; Mice, Knockout ; Cell Differentiation ; DNA-Binding Proteins/genetics* ; Mandibular Condyle/growth & development* ; Osteoblasts/cytology* ; Signal Transduction ; Neural Crest/cytology*

Objective: To provide high-quality clinical evidence of the efficacy of Tibetan medicine Honghua Ruyi (HHRY) pills for endometriosis-associated dysmenorrhea. Methods: This study constitutes a multicenter, randomized, double-blind, placebo-controlled trial encompassing a three-menstrual cycle intervention followed by a three-menstrual cycle follow-up period. A total of 164 eligible females with endometriosis-associated dysmenorrhea were randomly divided into HHRY pills and placebo groups in a 1:1 ratio. The primary outcome included dysmenorrhea symptoms assessed using Visual Analog Scale (VAS) scores and quality of life, whereas the secondary outcome measures included the maximum VAS for non-menstrual pelvic pain, duration of pain episodes (in days), frequency and quantity of the consumption of ibuprofen sustained-release capsules (or other non-steroidal anti-inflammatory drugs), and days off work/study for staff/student due to dysmenorrhea, ovarian cyst, and/or pelvic nodule size. The safety was monitored throughout the treatment period. All the analyses were based on the intention-to-treat principle. For continuous outcomes, simple or multiple linear regressions were used to estimate the differences between the HHRY pills and placebo groups, with categorical data expressed as the number and percentage of occurrences. Differences were compared using the chi-square test or Fisher's exact test. The predefined analysis was adjusted for concomitant treatment, a variable considered to be associated with outcomes but unaffected by treatment allocation. Estimates of treatment effects were reported with 95% confidence intervals. Two-tailed P values ≤ .05 were considered statistically significant. Conclusion Positive results from this trial, upon completion would provide robust evidence for the efficacy and safety of HHRY pills in treating dysmenorrhea in patients with endometriosis.

Objective To explore the mechanism of plumbagin for protecting against sepsis-induced myocardial injury in mice.Methods Network pharmacology analysis was used to obtain the key targets of plumbagin and diseases,which were subjected to GO and KEGG analysis,and the binding energy was verified using molecular docking.In a mouse model of cecal ligation and puncture(CLP),the protective effect of plumbagin treatment prior to CLP against sepsis-induced myocardial injury was evaluated by examination of myocardial function and pathology using echocardiography and HE staining.Serum levels of CK-MB,LDH,MDA,IL-1β and IL-18 and myocardial ROS level in the mice were detected,and Western blotting was used to determine the protein expression levels of STAT3,GSDMD,caspase-11,JAK2,P-STAT3,P-JAK2,GSDMD-N and HMGB1 in the myocardial tissues.Results Five core targets were screened from the 10 intersecting genes.Molecular docking showed strong binding affinity of plumbagin to STAT3,p-STAT3,and JAK2.Compared with the sham-operated mice,the mouse models of CLP-induced sepsis had significantly decreased CO,LVEF,LVFS and SV and increased serum levels of CK-MB,LDH,MDA and myocardial inflammatory factors and ROS.HE staining and Western blotting showed obvious myocardial injury in the septic mice with increased expressions of JAK2/STAT3 signaling pathway and pyroptosis-related proteins(P<0.05).Pretreatment with plumbagin significantly improved cardiac functions of CLP mice,lowered serum levels of CK-MB,LDH,MDA,inflammatory factors and myocardial ROS,and decreased the expression levels of JAK2/STAT3 signaling pathway and pyroptosis-related proteins.Conclusion Plumbagin pretreatment alleviates myocardial injury in septic mice possibly by inhibiting the STAT3 signaling pathway to reduce cardiomyocyte pyroptosis.

Objective To explore the mechanism of plumbagin for protecting against sepsis-induced myocardial injury in mice.Methods Network pharmacology analysis was used to obtain the key targets of plumbagin and diseases,which were subjected to GO and KEGG analysis,and the binding energy was verified using molecular docking.In a mouse model of cecal ligation and puncture(CLP),the protective effect of plumbagin treatment prior to CLP against sepsis-induced myocardial injury was evaluated by examination of myocardial function and pathology using echocardiography and HE staining.Serum levels of CK-MB,LDH,MDA,IL-1β and IL-18 and myocardial ROS level in the mice were detected,and Western blotting was used to determine the protein expression levels of STAT3,GSDMD,caspase-11,JAK2,P-STAT3,P-JAK2,GSDMD-N and HMGB1 in the myocardial tissues.Results Five core targets were screened from the 10 intersecting genes.Molecular docking showed strong binding affinity of plumbagin to STAT3,p-STAT3,and JAK2.Compared with the sham-operated mice,the mouse models of CLP-induced sepsis had significantly decreased CO,LVEF,LVFS and SV and increased serum levels of CK-MB,LDH,MDA and myocardial inflammatory factors and ROS.HE staining and Western blotting showed obvious myocardial injury in the septic mice with increased expressions of JAK2/STAT3 signaling pathway and pyroptosis-related proteins(P<0.05).Pretreatment with plumbagin significantly improved cardiac functions of CLP mice,lowered serum levels of CK-MB,LDH,MDA,inflammatory factors and myocardial ROS,and decreased the expression levels of JAK2/STAT3 signaling pathway and pyroptosis-related proteins.Conclusion Plumbagin pretreatment alleviates myocardial injury in septic mice possibly by inhibiting the STAT3 signaling pathway to reduce cardiomyocyte pyroptosis.

OBJECTIVE To investigate the effect of Huangqi-Ezhu-Chonglou combination on macrophage polarization and its mechanism of inhibiting colorectal cancer(CRC)cells proliferation and migration.METHODS THP-1 cells were stimulated with phorbol 12-myristate 13-acetate(PMA)and interleukin-4(IL-4)to establish M2 macrophage polarization model.The experiment was divided into M0 group(PMA treatment),M2 group(PMA+IL-4 treatment),and M2+ Huangqi-Ezhu-Chonglou combination group(PMA+IL-4+Huangqi-Ezhu-Chonglou combination treatment).The effect of Huangqi-Ezhu-Chonglou combination freeze-dried powder on the viability of macrophage was detected by CCK-8 method.The expression of macrophage polarization markers,glu-taminase(GLS)mRNA and protein was detected by qPCR and Western blot.The levels of interleukin-10(IL-10),transforming growth factor-β(TGF-β)and tumor necrosis factor-α(TNF-α)in cell supernatant were detected by ELISA.CCK-8 method and Tr-answell assays were used to detect the proliferation and migration of HCT116 cells intervened by the supernatant of macrophage culture treated with Huangqi-Ezhu-Chonglou combination,namely conditioned medium(CM).RESULTS Compared with the M0 group,the expression levels of IL-10,mannose receptor(CD206),arginase 1(ARG1),and GLS mRNA and protein in the M2 group were significantly increased(P<0.01,P<0.001),the levels of IL-10 and TGF-β secreted by macrophages were significantly increased(P<0.01,P<0.001);compared with the M2 group,the M2+ Huangqi-Ezhu-Chonglou combination group had significantly reduced IL-10,CD206,ARG1,and GLS mRNA and protein expression(P<0.05,P<0.01),the mRNA and protein levels of TNF-α and in-ducible nitric oxide synthase(iNOS)were significantly increased(P<0.05,P<0.01,P<0.001),the interleukin-1β(Interleukin-1β,IL-1β)mRNA expression significantly increased(P<0.01),and the contents of IL-10 and TGF-β in the cell supernatant sig-nificantly decreased(P<0.05,P<0.01),while TNF-α content significantly increased(P<0.01).CCK-8 and Transwell results showed that compared with the M0-CM group,the M2-CM promoted the proliferation and migration of HCT116 cells(P<0.01,P<0.001),the M2+ Huangqi-Ezhu-Chonglou-CM group significantly inhibited HCT116 cell proliferation and reduced cell migration compared to the M2-CM group(P<0.01,P<0.001).CONCLUSION Huangqi-Ezhu-Chonglou combination can inhibit colorectal cancer cells proliferation and migration by regulating macrophage polarization,and its mechanism may be related to the changes in the expression of GLS,a key enzyme in glutamine metabolism.

Objective To investigate the transcriptome differences of ovarian cancer cells after cisplatin(DDP)resistance,and to find potential antagonists based on this screening.Methods DDP-resistant cell line A2780-DDP was constructed with A2780 cells as the research object.Through transcriptome sequencing anal-ysis,the key factors of DDP resistance were found and verified by quantitative real-time PCR(qPCR)and Western blot experiments.Through the screening of small molecule inhibitors,CCK-8 cell viability assay was used to find potential antagonists.Results A2780-DDP were successfully constructed,and it was found that there was no difference in cell proliferation after drug resistance,but the ability of cell invasion and migration was enhanced.Through transcriptome sequencing analysis,it was found that ITGB7 and Akt may be the key genes of A2780-DDP,and qPCR and Western blot showed that they were highly expressed in A2780-DDP.CCK-8 results showed that triptolide(TPL)and Olaparib had good inhibitory effects in DDP-resistant cell lines.Conclusion The ITGB7/Akt pathway plays an important role in DDP resistance,and potential DDP re-sistance antagonists such as TPL can provide new ideas for the treatment of ovarian cancer.

Objective : By observing the changes of interleukin-22 ( IL-22) ，signal transduction and transcriptional activator 3 (STAT3) ，fasting blood glucose ( FBG) and fasting insulin ( FINS) of rats under the circumstance of chronic intermittent hypoxia and reoxygenation，to explore the role of IL-22 / STAT3 pathway in insulin resistance in- duced by chronic intermittent hypoxia． Methods : 4 SD rats were randomly divided into control group (NC group) and intermittent hypoxia group ( CIH group) ，with 12 rats in each group．NC group was placed in normoxia environment for 12 weeks，while CIH group was first given intermittent hypoxia for 8 weeks and then resumed normoxia feeding until 12 weeks．FBG，FINS，IL-22 and p-STAT3 / STAT3 levels were measured at baseline，week 8 and week 12 in both groups，and insulin resistance index (HOMA-IR) was calculated．The differences between the two groups were compared. Results : ① There was no significant difference of the observation indexes between the two groups at baseline (P＞0. 05) ．At 8 weeks，the levels of FBG，FINS and HOMA-IR in CIH group were higher than those in NC group (P＜0. 05) ，and the levels of IL-22 were lower than those in NC group (P ＜0. 05) ．p-STAT3 / STAT3 showed a decreasing trend，but not statistically significant．At 4 weeks of reoxygenation，there were no significant differences in FBG，FINS，HOMA-IR and IL-22 levels between the two groups (P ＞0. 05 ) ．p-STAT3 / STAT3 in CIH group was significantly higher than that in NC group ( P ＜0. 05 ) . ② Spearman rank correlation analysis showed that HOMA-IR was negatively correlated with IL-22 and p-STAT3 / STAT3 ( all P ＜0. 05) ． Conclusion Chronic intermittent hypoxia can inhibit the expression of IL-22 / STAT3 signaling pathway，IL-22 / STAT3 signaling pathway may mediate insulin resistance induced by chronic intermittent hypoxia.

Platycodon grandiflorus polysaccharide (PGP) is one of the main components of P. grandiflorus, but the mechanism of its anti-inflammatory effect has not been fully elucidated. The aim of this study was to evaluate the therapeutic effect of PGP on mice with dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) and explore the underlying mechanisms. The results showed that PGP treatment inhibited the weight loss of DSS-induced UC mice, increased colon length, and reduced DAI, spleen index, and pathological damage within the colon. PGP also reduced the levels of pro-inflammatory cytokines and inhibited the enhancement of oxidative stress and MPO activity. Meanwhile, PGP restored the levels of Th1, Th2, Th17, and Treg cell-related cytokines and transcription factors in the colon to regulate colonic immunity. Further studies revealed that PGP regulated the balance of colonic immune cells through mesenteric lymphatic circulation. Taken together, PGP exerts anti-inflammatory and anti-oxidant effect and regulates colonic immunity to attenuate DSS-induced UC through mesenteric lymphatic circulation.
Animals ; Mice ; Colitis, Ulcerative/drug therapy* ; Platycodon ; Colon/pathology* ; Cytokines ; Anti-Inflammatory Agents/therapeutic use* ; Polysaccharides/therapeutic use* ; Dextran Sulfate ; Disease Models, Animal ; Colitis/chemically induced* ; Mice, Inbred C57BL