1.Jieduan Niwan Formula regulates AMPK to alleviate mitochondrial damage in HepG2 cells
Ruimin MA ; Hanjing WANG ; Wenxin ZHANG ; Chongyang MA ; Qiuyun ZHANG ; Yuqiong DU
Journal of Beijing University of Traditional Chinese Medicine 2025;48(2):193-204
Objective:
To investigate the regulatory effect of Jieduan Niwan Formula (JDNWF) drug-containing serum on AMPK-mediated mitochondrial quality control in D-GalN-induced HepG2 cells.
Methods:
Twenty male Wistar rats were randomly divided into blank control and JDNWF-containing serum groups, 10 rats per group. The JDNWF-containing serum group was gavaged with JDNWF (21.7 g/kg), whereas the blank control group was gavaged with saline. Blood was collected to prepare JDNWF-containing and blank control serum. Cell viability, mitochondrial damage indicators, and MQC pathway protein expression levels were evaluated to determine the optimal volume fraction of JDNWF. HepG2 cells were divided into control, D-GalN, DMSO, AMPK inhibitor, JDNWF drug-containing serum, and JDNWF drug-containing serum plus AMPK inhibitor groups, and corresponding drug interventions were administered to each group. Cells were collected after the interventions, and the CCK-8 assay was used to measure cell viability, the 2′-7′-dichlorodihydrofluorescein diacetate fluorescent probe was used to detect reactive oxygen species (ROS) levels, JC-1 was used to detect mitochondrial membrane potential, thiobarbituric acid was used to measure malondialdehyde (MDA) levels, WST-8 was used to measure superoxide dismutase (SOD) activity, and western blotting was used to detect the expression levels of mitochondrial quality control-related proteins, including p-AMPK, AMPK, PGC-1α, NRF1, TFAM, MFN2, and DRP1.
Results:
5% JDNWF drug-containing serum most significantly restored cell viability, mitochondrial damage markers, and MQC pathway protein expression in the model group. Therefore, it was chosen for intervention in subsequent experiments. Compared to the control group, the cell viability of the D-GalN, DMSO, and AMPK inhibitor groups was significantly reduced (P<0.01). In contrast, the heterogeneity of mitochondrial membrane potential, ROS, and MDA levels was significantly increased (P<0.01), and SOD activity was significantly decreased (P<0.01). The p-AMPK, PGC-1α, NRF1, TFAM, MFN2, and DRP1 protein expression levels were significantly decreased (P<0.01). After JDNWF drug-containing serum intervention, compared to the DMSO group, cell viability significantly increased (P<0.01), mitochondrial membrane potential heterogeneity, ROS, and MDA levels significantly decreased (P<0.01), SOD activity significantly increased (P<0.01), and p-AMPK, PGC-1α, NRF1, TFAM, and MFN2 protein expression levels significantly increased (P<0.01), whereas DRP1 protein expression significantly decreased (P<0.01). Compared to the JDNWF drug-containing serum group, the cell viability in the JDNWF plus AMPK inhibitor group significantly decreased (P<0.01), mitochondrial membrane potential heterogeneity and ROS levels significantly increased (P<0.01), MDA levels significantly increased (P<0.05), SOD activity significantly decreased (P<0.05), p-AMPK, PGC-1α, NRF1, and TFAM protein expression levels significantly decreased (P<0.01), MFN2 protein expression significantly decreased (P<0.05), and DRP1 protein expression significantly increased (P<0.01).
Conclusion
JDNWF drug-containing serum may restore mitochondrial function and improve D-GalN-induced HepG2 cell injury by regulating AMPK-mediated mitochondrial quality control.
2.Comparison of three methods for induction of model mice with diminished ovarian reserve
Huadan LIN ; Xu WANG ; Chunhong CHEN ; Yuzhuo WEI ; Yanchun LUO ; Fanghui PANG ; Qiuyun QIN ; Ziyun RUAN
Chinese Journal of Comparative Medicine 2025;35(5):51-59
Objective To compare the effects of Tripterygium wilfordii polyglycosides,cyclophosphamide,and cisplatin on the establishment of a mouse model of diminished ovarian reserve(DOR).Methods Mice were randomly divided into the following treatment groups:control(Ctrl),Tripterygium wilfordii polyglycosides(TWP),cyclophosphamide(CTX),and cisplatin(DDP).Mice in the TWP group received a 50 mg/kg suspension of Tripterygium wilfordii polyglycosides by gavage for 14 days,mice in the CTX group received a 20 mg/kg cyclophosphamide suspension by intraperitoneal injection for 14 days,and mice in the DDP group received a 1.5 mg/kg cisplatin solution by intraperitoneal injection for 14 days.The body weight,uterine index,and ovarian index were recorded,the estrous cycle was monitored using the vaginal smear method,and the levels of anti-Mullerian hormone(AMH),estradiol(E2),follicle stimulating hormone(FSH),and luteinizing hormone(LH)were detected using ELISA.Hematoxylin and eosin staining was used to detect ovarian follicle development.The rates of oocyte maturation and fertility were analyzed.Results The three treatment groups of mice all showed the following:significantly decreased body weight and ovarian index(P<0.05);apparent disorder of the estrous cycle;significantly decreased levels of AMH and E2(P<0.05);decreased and increased rates of developing follicles and atretic follicles,respectively(P<0.05);and significantly decreased rates of oocyte maturation,pregnancy,and live birth(P<0.05).Conclusions DOR mouse models were successfully constructed using Tripterygium wilfordii polyglycosides,cyclophosphamide,or cisplatin,as evidenced by decreased body weight and ovarian index,disordered estrous cycle and hormones,and DOR function,resulting in reduced rates of oocyte maturation,pregnancy,and total number of live births.These DOR effects were most appropriate in the cyclophosphamide group.
3.Comparison of three methods for induction of model mice with diminished ovarian reserve
Huadan LIN ; Xu WANG ; Chunhong CHEN ; Yuzhuo WEI ; Yanchun LUO ; Fanghui PANG ; Qiuyun QIN ; Ziyun RUAN
Chinese Journal of Comparative Medicine 2025;35(5):51-59
Objective To compare the effects of Tripterygium wilfordii polyglycosides,cyclophosphamide,and cisplatin on the establishment of a mouse model of diminished ovarian reserve(DOR).Methods Mice were randomly divided into the following treatment groups:control(Ctrl),Tripterygium wilfordii polyglycosides(TWP),cyclophosphamide(CTX),and cisplatin(DDP).Mice in the TWP group received a 50 mg/kg suspension of Tripterygium wilfordii polyglycosides by gavage for 14 days,mice in the CTX group received a 20 mg/kg cyclophosphamide suspension by intraperitoneal injection for 14 days,and mice in the DDP group received a 1.5 mg/kg cisplatin solution by intraperitoneal injection for 14 days.The body weight,uterine index,and ovarian index were recorded,the estrous cycle was monitored using the vaginal smear method,and the levels of anti-Mullerian hormone(AMH),estradiol(E2),follicle stimulating hormone(FSH),and luteinizing hormone(LH)were detected using ELISA.Hematoxylin and eosin staining was used to detect ovarian follicle development.The rates of oocyte maturation and fertility were analyzed.Results The three treatment groups of mice all showed the following:significantly decreased body weight and ovarian index(P<0.05);apparent disorder of the estrous cycle;significantly decreased levels of AMH and E2(P<0.05);decreased and increased rates of developing follicles and atretic follicles,respectively(P<0.05);and significantly decreased rates of oocyte maturation,pregnancy,and live birth(P<0.05).Conclusions DOR mouse models were successfully constructed using Tripterygium wilfordii polyglycosides,cyclophosphamide,or cisplatin,as evidenced by decreased body weight and ovarian index,disordered estrous cycle and hormones,and DOR function,resulting in reduced rates of oocyte maturation,pregnancy,and total number of live births.These DOR effects were most appropriate in the cyclophosphamide group.
4.Clinical Study of Xuefu Zhuyu Decoction in the Prevention and Treatment of Radiation-Induced Lung Fi-brosis in Esophageal Cancer Patients with Blood Stasis Type Underwent Concurrent Chemoradiotherapy
Yijun WANG ; Lejun CHEN ; Jing YAN ; Teng HUANG ; Juan HAN ; Qiuyun YU ; Dahai YU ; Mianhua WU
Journal of Nanjing University of Traditional Chinese Medicine 2025;41(12):1657-1665
OBJECTIVE To observe the clinical efficacy of Xuefu Zhuyu Decoction(XFZY)in the prevention and treatment of radiation-induced lung fibrosis(RILF)in esophageal cancer patients with blood stasis type underwent concurrent chemoradiotherapy(CRT).METHODS A total of 130 esophageal cancer patients with blood stasis type who treated with concurrent CRT were randomly divided into an experimental group and a control group,with 65 cases in each group.No patients dropped out during the study period.Patients in both groups received CRT and standardized symptomatic treatment was given according to the condition if radiation-induced lung injury occurred during treatment.On the basis of the treatment of the control group,the patients in the experimental group received XFZY from the beginning day until 30 days after the completion of CRT.The TCM syndrome score of the two groups were compared before and after treatment.The incidence of acute radiation pneumonia(RP)and chronic RILF and changes in pulmo-nary function indicators[forced expiratory volume in the first second as a percentage of predicted value(FEV1%pred),forced vital ca-pacity(FVC),FVC as a percentage of predicted value(FVC%pred),FEV1/FVC ratio,and carbon monoxide diffusing capacity as a per-centage of predicted value(DLCO%pred)]and serum cytokine levels[interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),and transforming growth factor β1(TGF-β1)]were compared at 6 months and 12 months after the completion of CRT.The occurrence of adverse reaction during treatment was recorded.RESULTS The total score of TCM syndrome of the two groups was significantly improved and the experimental group was better than that of the control group(P<0.01)after treatment.The efficacy of TCM syndrome was better in the experimental group than that of the control group(P<0.01).There was no statistically significant difference in the incidence rate of acute RP between the two groups(P>0.05)at 6 months after the completion of CRT.The levels of lung function indicators FEV1%pred,FVC%pred,and DLCO%pred in the experimental group were higher than those in the control group(P<0.05),and the levels of various cytokines in the experimental group were lower than those in the control group(P<0.05).The incidence rate of chronic RILF in the experimental group was significantly lower than that of the control group(P<0.05)at 12 months after the completion of CRT.The DLCO%pred level in the experimental group was higher than that in the control group(P<0.01),and the levels of cytokines HIF-1α,VEGF,and TGF-β1 were lower than those in the control group(P<0.05,P<0.01).There was no serious adverse event observed in either group of patients during the treat-ment.CONCLUSION XFZY can effectively prevent and treat RILF in esophageal cancer patients with blood stasis type underwent CRT,reducing the loss to lung function caused by radiotherapy,and its mechanism may be related to downregulating the levels of cyto-kines of HIF-1α,VEGF,and TGF-β1.
5.Effect of subanesthetic dose of esketamine on emergence agitation in patients undergoing laparoscopic her-nia repair
Qiuyun WANG ; Mingcheng LI ; Huiyun GU ; Na TA ; Lige QI ; Lidong ZHU ; Jing GAO ; Xing-hua CAO
The Journal of Practical Medicine 2025;41(17):2728-2733
Objective To investigate the effect of subanesthetic dose of esketamine on emergence agitation(EA)in patients undergoing laparoscopic hernia repair.Methods Seventy-two male patients who treated with lapa-roscopic hernia repair under general anesthesia were randomly divided into AS group(subanesthetic esketamine)and control group.In the AS group,0.2 mg/kg of esketamine was administered intravenously 30 minutes before the surgery ended,while the control group was given an equal volume of normal saline.Upon surgery completion,patients were transferred to PACU with endotracheal tube retained,and the time to extubation was recorded.Hemo-dynamic parameters were measured immediately after extubation and at 10 min,30 min,1 h,and 4 h thereafter.Patients' pain and sedation levels were assessed at the above time points using RASS and VAS,respectively.The incidence of EA was evaluated using the Confusion Assessment Method for the Intensive Care Unit(CAM-ICU).Observation duration in the PACU and recovery outcomes within 24 hours postoperatively were assessed via the QoR-40 and adverse events were recorded.Results Compared to those in the control group,patients in the AS group had higher HR and MAP at 10 min post-extubation,and the changes in HR and MAP over time were more stable(P<0.05).The RASS and VAS scores in the AS group were significantly lower than those in the control group at the time of extubation and all subsequent time points(P<0.05),both groups showed temporal changes in RASS and VAS scores(P<0.05),but the change process in the AS group was more stable(P<0.05).Postoperative extubation time,PACU observation duration,and adverse event rates(delirium,respiratory depression,nausea and vomiting)did not differ significantly between the two groups(P>0.05),while recovery quality was markedly better in the AS group(P<0.05).Conclusion Subanesthetic esketamine effectively alleviates pain and the incidence of EA,supports hemodynamic stability during PACU stay,and enhances recovery quality in patients undergoing laparo-scopic hernia repair,demonstrating clinical value.
6.Clinical Study of Xuefu Zhuyu Decoction in the Prevention and Treatment of Radiation-Induced Lung Fi-brosis in Esophageal Cancer Patients with Blood Stasis Type Underwent Concurrent Chemoradiotherapy
Yijun WANG ; Lejun CHEN ; Jing YAN ; Teng HUANG ; Juan HAN ; Qiuyun YU ; Dahai YU ; Mianhua WU
Journal of Nanjing University of Traditional Chinese Medicine 2025;41(12):1657-1665
OBJECTIVE To observe the clinical efficacy of Xuefu Zhuyu Decoction(XFZY)in the prevention and treatment of radiation-induced lung fibrosis(RILF)in esophageal cancer patients with blood stasis type underwent concurrent chemoradiotherapy(CRT).METHODS A total of 130 esophageal cancer patients with blood stasis type who treated with concurrent CRT were randomly divided into an experimental group and a control group,with 65 cases in each group.No patients dropped out during the study period.Patients in both groups received CRT and standardized symptomatic treatment was given according to the condition if radiation-induced lung injury occurred during treatment.On the basis of the treatment of the control group,the patients in the experimental group received XFZY from the beginning day until 30 days after the completion of CRT.The TCM syndrome score of the two groups were compared before and after treatment.The incidence of acute radiation pneumonia(RP)and chronic RILF and changes in pulmo-nary function indicators[forced expiratory volume in the first second as a percentage of predicted value(FEV1%pred),forced vital ca-pacity(FVC),FVC as a percentage of predicted value(FVC%pred),FEV1/FVC ratio,and carbon monoxide diffusing capacity as a per-centage of predicted value(DLCO%pred)]and serum cytokine levels[interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),and transforming growth factor β1(TGF-β1)]were compared at 6 months and 12 months after the completion of CRT.The occurrence of adverse reaction during treatment was recorded.RESULTS The total score of TCM syndrome of the two groups was significantly improved and the experimental group was better than that of the control group(P<0.01)after treatment.The efficacy of TCM syndrome was better in the experimental group than that of the control group(P<0.01).There was no statistically significant difference in the incidence rate of acute RP between the two groups(P>0.05)at 6 months after the completion of CRT.The levels of lung function indicators FEV1%pred,FVC%pred,and DLCO%pred in the experimental group were higher than those in the control group(P<0.05),and the levels of various cytokines in the experimental group were lower than those in the control group(P<0.05).The incidence rate of chronic RILF in the experimental group was significantly lower than that of the control group(P<0.05)at 12 months after the completion of CRT.The DLCO%pred level in the experimental group was higher than that in the control group(P<0.01),and the levels of cytokines HIF-1α,VEGF,and TGF-β1 were lower than those in the control group(P<0.05,P<0.01).There was no serious adverse event observed in either group of patients during the treat-ment.CONCLUSION XFZY can effectively prevent and treat RILF in esophageal cancer patients with blood stasis type underwent CRT,reducing the loss to lung function caused by radiotherapy,and its mechanism may be related to downregulating the levels of cyto-kines of HIF-1α,VEGF,and TGF-β1.
7.Analysis of imaging signs of ovarian torsion in children
Hui MA ; Lihua DAI ; Yingfei WANG ; Jianjun LIN ; Qiuyun TANG ; Daxing XU
Journal of Practical Radiology 2025;41(2):285-288
Objective To summarize and analyze the CT or MRI imaging signs of ovarian torsion in children.Methods The CT or MRI data of 24 children surgically confirmed ovarian torsion were analyzed retrospectively,focusing on imaging signs such as ovarian position,size,the relationship with surrounding appendages and uterus.Results In this group of cases,8 cases underwent CT examination and 16 cases underwent MRI examination.Among the age of children,12 cases were in newborns,and 6 cases were in school-age and adolescent children respectively.It was more common in newborns and children aged 10-12 years old.Among the 24 patients,3 cases(12.5%)had primary ovarian torsion,all of which were adolescent children;21 cases(87.5%)had secondary ovarian torsion,with all torsions in the neonatal period were secondary ovarian torsion.Among secondary ovarian torsion,there were 7 cases(33.3%)of ovarian teratoma with torsion,12 cases(57.1%)of simple ovarian cysts,1 case(4.7%)of ovarian serous cystadenoma,and 1 case(4.7%)of ovarian mucinous cyst.Torsion occurred in 9 cases(37.5%)of the left ovary and 15 cases(62.5%)of the right ovary,with right ovarian torsion being more common.Imaging signs included varying degrees of enlargement of the ovaries on the ipsilateral side of the torsion,with 14 cases(58.3%)of ovarian masses had a maximum diameter≥5 cm,and 3-5 cm being more common in the neonatal period.There were 11 cases(45.8%)with the pedicle sign/vortex sign on the ipsilateral side of the torsion,9 cases(37.5%)with mass and hemorrhage,4 cases(16.7%)with mass displacement to the midline or uterine displacement to the ipsilateral side,and 3 cases(12.5%)of the ovarian follicle outward migration showed fruit bowl sign.Conclusion Secondary ovarian torsion is relatively common in children,with distinctive imaging manifestations.Especially when neonatal ovarian cysts show hemorrhagic signals should be alert to the risk of ovarian torsion.CT and MRI examinations can provide a powerful reference for the clinical diagnosis of ovarian torsion in children.
8.Effect of subanesthetic dose of esketamine on emergence agitation in patients undergoing laparoscopic her-nia repair
Qiuyun WANG ; Mingcheng LI ; Huiyun GU ; Na TA ; Lige QI ; Lidong ZHU ; Jing GAO ; Xing-hua CAO
The Journal of Practical Medicine 2025;41(17):2728-2733
Objective To investigate the effect of subanesthetic dose of esketamine on emergence agitation(EA)in patients undergoing laparoscopic hernia repair.Methods Seventy-two male patients who treated with lapa-roscopic hernia repair under general anesthesia were randomly divided into AS group(subanesthetic esketamine)and control group.In the AS group,0.2 mg/kg of esketamine was administered intravenously 30 minutes before the surgery ended,while the control group was given an equal volume of normal saline.Upon surgery completion,patients were transferred to PACU with endotracheal tube retained,and the time to extubation was recorded.Hemo-dynamic parameters were measured immediately after extubation and at 10 min,30 min,1 h,and 4 h thereafter.Patients' pain and sedation levels were assessed at the above time points using RASS and VAS,respectively.The incidence of EA was evaluated using the Confusion Assessment Method for the Intensive Care Unit(CAM-ICU).Observation duration in the PACU and recovery outcomes within 24 hours postoperatively were assessed via the QoR-40 and adverse events were recorded.Results Compared to those in the control group,patients in the AS group had higher HR and MAP at 10 min post-extubation,and the changes in HR and MAP over time were more stable(P<0.05).The RASS and VAS scores in the AS group were significantly lower than those in the control group at the time of extubation and all subsequent time points(P<0.05),both groups showed temporal changes in RASS and VAS scores(P<0.05),but the change process in the AS group was more stable(P<0.05).Postoperative extubation time,PACU observation duration,and adverse event rates(delirium,respiratory depression,nausea and vomiting)did not differ significantly between the two groups(P>0.05),while recovery quality was markedly better in the AS group(P<0.05).Conclusion Subanesthetic esketamine effectively alleviates pain and the incidence of EA,supports hemodynamic stability during PACU stay,and enhances recovery quality in patients undergoing laparo-scopic hernia repair,demonstrating clinical value.
9.Analysis of imaging signs of ovarian torsion in children
Hui MA ; Lihua DAI ; Yingfei WANG ; Jianjun LIN ; Qiuyun TANG ; Daxing XU
Journal of Practical Radiology 2025;41(2):285-288
Objective To summarize and analyze the CT or MRI imaging signs of ovarian torsion in children.Methods The CT or MRI data of 24 children surgically confirmed ovarian torsion were analyzed retrospectively,focusing on imaging signs such as ovarian position,size,the relationship with surrounding appendages and uterus.Results In this group of cases,8 cases underwent CT examination and 16 cases underwent MRI examination.Among the age of children,12 cases were in newborns,and 6 cases were in school-age and adolescent children respectively.It was more common in newborns and children aged 10-12 years old.Among the 24 patients,3 cases(12.5%)had primary ovarian torsion,all of which were adolescent children;21 cases(87.5%)had secondary ovarian torsion,with all torsions in the neonatal period were secondary ovarian torsion.Among secondary ovarian torsion,there were 7 cases(33.3%)of ovarian teratoma with torsion,12 cases(57.1%)of simple ovarian cysts,1 case(4.7%)of ovarian serous cystadenoma,and 1 case(4.7%)of ovarian mucinous cyst.Torsion occurred in 9 cases(37.5%)of the left ovary and 15 cases(62.5%)of the right ovary,with right ovarian torsion being more common.Imaging signs included varying degrees of enlargement of the ovaries on the ipsilateral side of the torsion,with 14 cases(58.3%)of ovarian masses had a maximum diameter≥5 cm,and 3-5 cm being more common in the neonatal period.There were 11 cases(45.8%)with the pedicle sign/vortex sign on the ipsilateral side of the torsion,9 cases(37.5%)with mass and hemorrhage,4 cases(16.7%)with mass displacement to the midline or uterine displacement to the ipsilateral side,and 3 cases(12.5%)of the ovarian follicle outward migration showed fruit bowl sign.Conclusion Secondary ovarian torsion is relatively common in children,with distinctive imaging manifestations.Especially when neonatal ovarian cysts show hemorrhagic signals should be alert to the risk of ovarian torsion.CT and MRI examinations can provide a powerful reference for the clinical diagnosis of ovarian torsion in children.
10.Cuiru Keli Improves Postpartum Hypogalactia in Rats Through Secreted Frizzled-Related Protein 2-Wnt/β-catenin Signaling Pathway
Qiuyun XUE ; Yurong HUANG ; Hui LI ; Chen LI ; Chenglong CHENG ; Yuting WANG ; Chenggui MIAO
Journal of Sichuan University (Medical Sciences) 2024;55(3):619-629
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.


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