ObjectiveTo observe ovarian microvascular damage in rats with cold coagulation and blood stasis syndrome and to explore the mechanism by which Liangfang Wenjing decoction improves this condition in rats. MethodsFifty SPF female SD rats were randomly divided into a blank group， a model group， low-dose （8.1 g·kg^-1） and high-dose groups （16.2 g·kg^-1） of Liangfang Wenjing decoction， and a 4-phenylbutyric acid （0.1 g·kg^-1） group， with 10 rats in each group. The ice-water bath method was employed to establish the rat model of cold coagulation and blood stasis syndrome. Concurrent with modeling， Liangfang Wenjing decoction was administered continuously for 21 days， once daily. The rats' syndrome manifestations and estrous cycles were recorded. The enzyme-linked immunosorbent assay （ELISA） was used to detect serum reproductive hormone levels and levels of endothelin-1 （ET-1）， nitric oxide （NO）， thrombomodulin （TM）， and von Willebrand factor （vWF） in ovarian tissue. Prothrombin time （PT）， activated partial thromboplastin time （APTT）， thrombin time （TT）， and fibrinogen （FIB） were measured. The ovarian microcirculatory blood perfusion was detected by laser speckle contrast imaging. Hematoxylin-eosin （HE） staining was performed to observe the ovarian histopathology， flow cytometry to detect ovarian apoptosis rate， and transmission electron microscopy to observe the ultrastructure of ovarian microvascular endothelial cells. Western blot was employed to detect the protein expression of endothelial nitric oxide synthase （eNOS）， phosphorylated eNOS （p-eNOS）， Caspase-3， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， glucose-regulated protein 78 （GRP78）， C/EBP homologous protein （CHOP）， inositol-requiring enzyme1α （IRE1α）， p-IRE1α， apoptosis signal-regulating kinase 1 （ASK1）， p-ASK1， c-Jun N-terminal kinase （JNK）， and p-JNK. Immunofluorescence was used to detect ovarian Bax and Bcl-2 expression in microvascular endothelial cells. ResultsCompared with the blank group， the model group showed signs of cold coagulation and blood stasis syndrome， prolonged estrus cycles， and reproductive hormone disorders. Histopathological results revealed a decrease in follicle counts at all stages and disorganized granulosa cell arrangement. Ovarian microcirculatory perfusion was significantly decreased （P<0.01）. PT， APTT， and TT were reduced （P<0.05， P<0.01）， while FIB levels were increased （P<0.05）. In ovarian tissue， NO content was decreased， while ET-1， vWF， and TM levels were increased significantly （P<0.01）. The apoptosis rate of ovarian cells was markedly increased （P<0.01）. Furthermore， p-eNOS/eNOS and Bcl-2 were decreased （P<0.05）， whereas Bax， cleaved-Caspase-3/Caspase-3， GRP78， CHOP， p-IRE1α/IRE1α， p-ASK1/ASK1， and p-JNK/JNK expression showed significant increases （P<0.05， P<0.01）. Compared with the model group， Liangfang Wenjing decoction intervention alleviated the symptoms of cold coagulation and blood stasis， gradually restored the estrus cycle， and improved ovarian histopathology and endothelial cell ultrastructure. Microcirculatory blood perfusion was significantly elevated （P<0.05）. NO content in ovarian tissue was elevated， while ET-1， vWF， and TM levels were significantly decreased （P<0.05， P<0.01）. The p-eNOS/eNOS ratio and Bcl-2 expression were significantly elevated （P<0.05）， while the expression of Bax， cleaved-Caspase-3/Caspase-3， GRP78， CHOP， p-IRE1α/IRE1α， p-ASK1/ASK1， and p-JNK/JNK was significantly decreased （P<0.05， P<0.01）. ConclusionLiangfang Wenjing decoction may regulate the IRE1α/ASK1/JNK signaling pathway to inhibit endoplasmic reticulum stress， attenuate apoptosis， and improve microvascular endothelial injury in ovaries of rats with cold coagulation and blood stasis syndrome.

Background and purpose:The overexpression of carnitine palmitoyl transferase 1A(CPT1A)is related to the poor prognosis of breast cancer,and it can promote the utilization of fatty acids by mitochondria and maximize triphosphate(ATP)production.However,the role of CPT1A in breast cancer metastasis is still unclear.This study aimed to explore the mechanism that mitochondrial dysfunction and CPT1A/extracellular signal-regulated kinase(ERK)signaling pathway in breast cancer jointly regulate the malignant behavior of breast cancer.Methods:The lentivirus system and shRNA tools were used to overexpress or knock down CPT1A in human breast cancer cell lines MDA-MB-231 and MCF7,and the cells were divided into NC group,CPT1A group and shCPT1A group.The invasion ability of cells was detected by transwell assay,and the protein expressions of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α),ERK1/2 and CPT1A were analyzed by Western blot.The mitochondrial morphology of MDA-MB-231 and MCF7 cell lines was analyzed using mitochondrial red staining,and mitochondrial respiratory capacity was analyzed by oxygen consumption rate.Results:Compared with cells expressing control vector,overexpression of CPT1A resulted in enhanced invasion abilities of MCF7 cells and MDA-MB-231 cells(P＜0.05),while knockdown of shCPT1A resulted in decreased invasion abilities of cells(P＜0.05).Compared with NC group,the length of mitochondrial branches in MDA-MB-231 and MCF7 cells in CPT1A group was significantly shorter(P＜0.05),and the expressions of ERK1/2 and PGC-1α increased significantly(P＜0.05).In shCPT1A group,the length of mitochondrial branches in MDA-MB-231 and MCF7 cells increased significantly(P＜0.05),and the expressions of ERK1/2 and PGC-1α decreased significantly(P＜0.05).In addition,compared with NC group,the cellular basis,maximum respiratory capacity and ATP production of MDA-MB-231 in CPT1A group increased significantly(P＜0.05),while the cellular basis,maximum respiratory capacity and ATP production of MDA-MB-231 in shCPT1A group decreased significantly(P＜0.05).Conclusion:ERK1/2-PGC-1α activated by CPT1A Signaling pathway plays a key role in mitochondrial division mediated breast cancer cell metastasis.

Objective:To seek specific response points on the body surface of patients with primary dysmenorrhea(PD)by observing blood perfusion unit(PU)at different points of the three Yin meridians of foot using laser speckle contrast imaging(LSCI). Methods:Eighty PD patients were recruited as a PD group,and 80 healthy female undergraduates were taken as a normal group.During one menstrual cycle(before menstruation,during menstruation,and 3 d after menstruation),each participant was examined using the LSCI system to determine PU at bilateral Taixi(KI3),Taibai(SP3),Taichong(LR3),Shuiquan(KI5),Diji(SP8),Zhongdu(LR6),Sanyinjiao(SP6),and Xuehai(SP10)and non-acupuncture points.The researchers in charge of point location,operation,and statistical analysis were not aware of grouping.PU at the detection spots was taken as the outcome measure. Results:Compared with the normal group,the PD group showed increases in PU at right Taixi(KI3)before menstruation(P<0.05)and at bilateral Zhongdu(LR6)and right Diji(SP8)during menstruation(P<0.05).At the other time points,significance was not found between the two groups in comparing PU at the detected spots. Conclusion:Compared with healthy participants,PD patients present specific changes in PU at Taixi(KI3),Diji(SP8),and Zhongdu(LR6)at specific time points during the menstrual cycle,which provides a reference for acupuncture-moxibustion treatment of PD in clinical settings.

Objective: To study the effect of miR-28-5p on cisplatin ( DDP) -resistant A549 lung adenocarcinoma cell line (A549 / DDP) ，and to explore whether its mechanism is related to ferroptosis suppressor protein 1 (FSP1) mediated ferroptosis． Methods: The A549 lung adenocarcinoma cell line and A549 / DDP cells were selected as the research objects. RT-qPCR method was used to detect the expression level of miR-28-5p in the cells．Cell proliferation was measured by CCK-8 method and colony formation assay．The target gene of miR-28-5p was identified and verified by luciferase reporter gene and Western blot analysis．A549 / DDP was transfected with miR-28-5p simulant or FSP1 overexpression plasmid to evaluate cell proliferation，mitochondrial morphology was evaluated by transmission electron microscope，and the levels of reactive oxygen species (ROS) ，glutathione ( GSH) and malondialdehyde (MDA) were measured by kit． A cell line-based xenograft model was established to evaluate the effect of miR-28-5p on tumor growth in vivo. Results: Compared with A549 cells ，the expression level of miR-28-5p in A549 / DDP cells was significantly reduced ( P ＜0. 001 ) ． Compared with A549 cells ，the cell viability ( F = 49. 542，P＜0. 001) and colony forming ability (t = 4. 412，P＜0. 01) of A549 / DDP cells increased significantly. Compared with miR-NC group，the cell viability (t = 4. 612，P＜0. 01) and colony number (t = 4. 503，P＜0. 01) of A549 / DDP cells in miR-28-5p group significantly decreased．The luciferase activity decreased in the cells transfected with the miR-28-5p mimic，being significantly more so in the presence of the pGL3-FSP1 3 'UTR-WT vector. In addition，the expression of FSP1 in cells overexpressing miR-28-5p was significantly suppressed．Compared with the Vector + miR-28-5p group，the FSP1 + miR-28-5p group significantly increased cell viability and colony formation，cell mitochondrial length and GSH (P ＜0．01) ，and significantly increased cell apoptosis，ROS production and MDA formation decrease (P ＜0. 05 ) ． In vivo experiments showed that compared with the miR-NC + DDP group，the size and weight of tumors formed in the miR-28-5p + DDP group were significantly reduced (P＜0. 05) ， and the expression of Ki-67 and FSP1 protein was significantly reduced (P＜0. 05) ． Conclusion The mechanism of miR-28-5p reversing cisplatin resistance in lung adenocarcinoma cells may be related to the inhibition of FSP1 mediated ferroptosis.

Objective:To evaluate whether FOXO4-DRI could reverse radiation-induced pulmonary fibrosis (RIPF) and to explore the underlying mechanism.Methods:C57BL/6 mice were randomly divided into 4 groups: control, FOXO4-DRI, radiation, and radiation+ FOXO4-DRI. Mice in radiation or radiation+ FOXO4-DRI groups received 17 Gy X-ray radiation on the right side of the whole chest. Mice in FOXO4-DRI and radiation+ FOXO4-DRI groups were injected with FOXO4-DRI intraperitoneally at 16 and 20 weeks after irradiation, respectively. The right lungs were collected at 24 weeks after irradiation and subjected to HE staining and Masson trichrome staining to observe the morphological changes and collagen deposition. Immunohistochemistry was used to evaluate the expressions of col1α1 and α-SMA in lung tissues. β-gal staining was used to observe senescent cells. The level of reactive oxygen species in lung tissue was detected. The expressions of P21, P16 Ink4a and senescence-associated secretory phenotype (SASP) mRNA were detected by qRT-PCR, and the expression of related proteins were assessed by Western blot. Results:FOXO4-DRI reduced collagen deposition ( t=6.18, P<0.05), down-regulated the expression of col1α1 and α-SMA ( t=4.69, 3.20, P<0.05), and reduced the number of β-gal positive cells ( t=6.09, P<0.05) in the lung tissue of RIPF mice. FOXO4-DRI also down-regulated the gene and protein expressions of P21 and P16 Ink4a ( t=5.31, 3.32 and 4.77, 3.37, P<0.05) and inhibited the expressions of SASP genes IL-1α, IL-1β, TNF-α and MMP2 ( t=4.36, 4.84, 4.47, 3.82, P<0.05), reduced reactive oxygen species ( t=2.84, P<0.05), and promoted the activation of p-AKT and p-PI3K proteins ( t=-7.13, -12.61, P< 0.05) in the lung tissue of RIPF mice. Conclusions:FOXO4-DRI reverses RIPF by activating the PI3K/AKT signaling pathway, reducing oxidative stress and inhibiting cellular senescence.

The cold congeal and blood stasis syndrome is a common clinical traditional Chinese medicine（TCM） syndrome. The animal model of cold ongeal and blood stasis syndrome is the basis for exploring the essence of TCM cold congeal and blood stasis syndrome，and the premise of follow-up TCM clinical research.This paper summarized the preparation method， theoretical support，and evaluation method of animal models of cold congeal and blood stasis syndrome in recent years and analysed the strengthens and weaknesses of different models. At present，the common animal models of cold congeal and blood stasis syndrome mainly include etiological model，etiological and pathological composite model and disease-syndrome combination model. The etiological model was mainly prepared by cold exposure，which could be divided into whole-body freezing， ice bath and local frostbite. The etiological and pathological composite model was mainly prepared by cold stimulation combined with epinephrine injection. The common disease-syndrome combination models included the coronary heart disease model of cold congeal and blood stasis syndrome，primary dysmenorrhea model of cold congeal and blood stasis syndrome，endometriosis model of cold congeal and blood stasis syndrome， and arteriosclerosis obliterans model of cold congeal and blood stasis syndrome. The three models have both advantages and disadvantages. Specifically， the disease-syndrome combination model had the highest consistency with clinical practice and was more reliable and practical. However， the disease types of this model were specific，and the combination method of disease and syndrome was controversial. The evaluation indicators of the animal models of cold congeal and blood stasis syndrome focused on the characterization of the syndrome and the physico-chemical indicators related to blood flow，such as blood rheology，coagulation function and microcirculation. In addition， some scholars explored the evaluation indicators from the aspects of vasomotor function，endocrine and energy metabolism. The objectivity and specificity of the current model evaluation methods needed to be further improved. The research of animal model of cold congeal and blood stasis syndrome should be based on clinical practice and oriented by clinical demand. Only by establishing animal models that are highly consistent with the characteristics of clinical disease and syndrome can we better reveal the essence of cold congeal and blood stasis syndrome and promote the modernization of TCM.

Objective:To investigate the correlation between serum uric acid level and symptomatic intracranial hemorrhage (sICH) and outcomes after endovascular therapy (EVT) in elderly patients with anterior circulation acute ischemic stroke (AIS).Methods:Elderly patients with AIS (aged ≥65 years) received EVT in Beijing Geriatric Hospital and Xuanwu Hospital from December 2017 to December 2020 were retrospectively enrolled. sICH was defined as cerebral parenchymal hemorrhage revealed by CT within 72 h after admission and the Naitonal Institutes of Health Stroke Scale score increased by ≥4 compared with the baseline. At 90 d after onset, the clinical outcome was evaluated by the modified Rankin Scale. 0-2 was a good outcome and 3-6 was a poor outcome. The clinical data of the sICH group and non-sICH group, as well as the good outcome group and poor outcome group were compared. Multivariate logistic regression analysis was used to determine the independent correlation between serum uric acid level and sICH and poor outcomes. Results:A total of 122 patients were enrolled, their age was 73.89±6.24 years, and 73 (59.8%) were male. Fifty-two patients (42.6%) had hemorrhagic transformation, 27 (22.1%) had sICH, and 28 (23.8%) had a good outcome at 90 d after onset. The serum uric acid in the sICH group was significantly lower than that in the non-sICH group ( P=0.002), while the serum uric acid in the good outcome group was similar to that in the poor outcome group ( P=0.510). Multivariate logistic analysis showed that the lower serum uric acid was an independent risk factor for sICH (odds ratio 0.994, 95% confidence interval 0.990-0.998; P=0.011). Conclusion:The lower serum uric acid level was an independent risk factor for sICH after EVT in elderly patients with AIS, but it was not associated with the outcomes.

Objective:To evaluate the prognosis and determine the failure patterns after radiotherapy for low-risk early-stage patients with extranodal NK/T-cell lymphoma, nasal-type (ENKTCL).Methods:A total of 557 patients from 2000—2015 with low-risk early-stage ENKTCL who received radiotherapy (RT) with or without chemotherapy (CT) from China Lymphoma Collaborative Group were retrospectively reviewed. Among them, 427 patients received combined modality therapy, whereas 130 patients received RT alone. Survivals were calculated by Kaplan-Meier method and compared with Log-rank test. Overall survival (OS) was compared with age and sex-matched general Chinese population using expected survival and standardized mortality ratio (SMR). Cox stepwise regression model was used for multivariate analysis.Results:The 5-year OS and progression-free survival (PFS) were 87.2% and 77.2%. The SMR was 3.59 ( P<0.001) at 1 year after treatment, whereas it was 1.50 at 4 years after treatment, without significant difference between ENKTCL group and country-matched general population ( P=0.146). Compared with RT alone, CMT did not result in significantly superior 5-year OS (87.0% vs 87.4%, P=0.961) or PFS (76.1% vs 80.7%, P=0.129). Local failure (11.5%, 64/557) and distant failure (10.8%, 60/557) were the main failure modes, while regional failure was rare (2.9%, 16/557). The 5-year locoregional control rate (LRC) was 87.2% for the whole group, with 89.5% for ≥50 Gy versus 73.7% for <50 Gy ( P<0.001). Radiotherapy dose was an independent factor affecting LRC( P<0.05). Conclusions:Radiotherapy achieves a favorable prognosis in patients with low-risk early-stage ENKTCL. The incidence of either locoregional or distant failure is low. Radiation dose still is an important prognostic factor for LRC.

Objective:To evaluate the prognosis and determine the failure patterns after radiotherapy for low-risk early-stage patients with extranodal NK/T-cell lymphoma, nasal-type (ENKTCL).Methods:A total of 557 patients from 2000—2015 with low-risk early-stage ENKTCL who received radiotherapy (RT) with or without chemotherapy (CT) from China Lymphoma Collaborative Group were retrospectively reviewed. Among them, 427 patients received combined modality therapy, whereas 130 patients received RT alone. Survivals were calculated by Kaplan-Meier method and compared with Log-rank test. Overall survival (OS) was compared with age and sex-matched general Chinese population using expected survival and standardized mortality ratio (SMR). Cox stepwise regression model was used for multivariate analysis.Results:The 5-year OS and progression-free survival (PFS) were 87.2% and 77.2%. The SMR was 3.59 ( P<0.001) at 1 year after treatment, whereas it was 1.50 at 4 years after treatment, without significant difference between ENKTCL group and country-matched general population ( P=0.146). Compared with RT alone, CMT did not result in significantly superior 5-year OS (87.0% vs 87.4%, P=0.961) or PFS (76.1% vs 80.7%, P=0.129). Local failure (11.5%, 64/557) and distant failure (10.8%, 60/557) were the main failure modes, while regional failure was rare (2.9%, 16/557). The 5-year locoregional control rate (LRC) was 87.2% for the whole group, with 89.5% for ≥50 Gy versus 73.7% for <50 Gy ( P<0.001). Radiotherapy dose was an independent factor affecting LRC( P<0.05). Conclusions:Radiotherapy achieves a favorable prognosis in patients with low-risk early-stage ENKTCL. The incidence of either locoregional or distant failure is low. Radiation dose still is an important prognostic factor for LRC.

OBJECTIVE: To explore the molecular basis for an individual with postnatal deafness and provide genetic counseling for her family. METHODS: Following extraction of genomic DNA from peripheral blood samples, 127 genes associated with deafness were subjected to targeted capturing and next generation sequencing. Suspected mutation was verified by Sanger sequencing. RESULTS: The proband was found to carry a homozygous c.1893C>A mutation in the TECTA gene, which is located in the tectorial membrane of inner ear and may cause premature termination of translation of TECTA protein. In addition, two heterozygous mutations, c.13010C>T and c.12790G>A, were found in the USH2A gene. Whilst the former is likely to be pathogenic, the latter has unknown clinical significance. Further analysis suggested that all three mutations have derived from the parents of the proband. CONCLUSION The homozygous c.1893C>A mutation of the TECTA gene probably underlies the proband's hearing loss which conformed to an autosomal recessive inheritance.
Deafness ; Extracellular Matrix Proteins ; genetics ; Female ; GPI-Linked Proteins ; genetics ; High-Throughput Nucleotide Sequencing ; Homozygote ; Humans ; Mutation ; Pedigree