ObjectiveTo clarify the neuroprotective effect of black Panacis Quinquefolii Radix（PQR） and explore its active ingredients， with the aim of establishing an activity-oriented quality evaluation method. MethodsTransgenic Tg（HuC∶EGFP） zebrafish was used to establish a neuronal injury model by aluminum chloride immersion. Different doses（10， 20 mg·L^-1） of PQR and black PQR ethanol extracts were administered. The neuroprotective effects of PQR and black PQR were compared by analyzing the fluorescent area and intensity of zebrafish neurons. Based on ultra-performance liquid chromatography（UPLC）， a fingerprint profile of black PQR was established， followed by principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）. Differential components were screened using the criteria of variable importance in the projection（VIP） value>1 and P<0.05. The neuroprotective activity of 14 batches of black PQR was assessed， and Spearman correlation analysis was used to identify saponins related to neuroprotective activity， which were then validated. Based on the above results， active marker components were determined， and an UPLC method was established for their quantitation with clear content limits. ResultsPharmacological efficacy results showed that both PQR and black PQR at different doses could significantly improved neuronal damage in zebrafish. At a dose of 20 mg·L^-1， black PQR demonstrated superior efficacy（P<0.05）. The fingerprint similarities of 14 batches of black PQR were>0.94， with 26 common peaks identified. Through comparison with the reference standards， 8 components were confirmed， including peak 1（ginsenoside Rg₁）， peak 2（ginsenoside Re）， peak 5（ginsenoside Rb₁）， peak 9（ginsenoside Rd）， peak 16［ginsenoside 20（S）-Rg₃］， peak 17［ginsenoside 20（R）-Rg₃］， peak 18（ginsenoside Rk₁）， and peak 19（ginsenoside Rg₅）. The results of PCA and OPLS-DA indicated that there were differences in saponins among black PQR samples from different origins， and 12 differential components were screened. All 14 batches of black PQR exhibited good protective effects on zebrafish neurons， with Shaanxi-produced black PQR showing superior protective effects compared to the other three production regions. Spearman correlation analysis revealed that a total of 11 components， including ginsenosides 20（S）-Rg₃， 20（R）-Rg₃， Rk₁ and Rg₅， showed a significant positive correlation with the neuroprotective effect in zebrafish（P<0.05）. The activity validation results indicated that ginsenosides 20（S）-Rg₃， 20（R）-Rg₃， Rk₁ and Rg₅ were the primary components responsible for the neuroprotective effects of black PQR. Quantitative analysis showed that the content of ginsenoside 20（S）-Rg₃ in 14 batches of black PQR ranged from 0.17% to 0.52%， and the repair rate of neuronal damage ranged from 42.77% to 97.83%. ConclusionBased on the fingerprint and neuronal protective activity， the spectrum-effect related quality control model of black PQR was established， with ginsenoside 20（S）-Rg₃ as the quality control index， and the neuronal damage repair rate≥60% as the evaluation standard， the minimum limit of ginsenoside 20（S）-Rg₃ in black PQR should be≥0.20%.

Articular cartilage (AC) injuries often lead to cartilage degeneration and may ultimately result in osteoarthritis (OA) due to the limited self-repair ability. To date, numerous intra-articular delivery systems carrying various therapeutic agents have been developed to improve therapeutic localization and retention, optimize controlled drug release profiles and target different pathological processes. Due to the complex and multifactorial characteristics of cartilage injury pathology and heterogeneity of the cartilage structure deposited within a dense matrix, delivery systems loaded with a single therapeutic agent are hindered from reaching multiple targets in a spatiotemporal matched manner and thus fail to mimic the natural processes of biosynthesis, compromising the goal of full cartilage regeneration. Emerging evidence highlights the importance of sequential delivery strategies targeting multiple pathological processes. In this review, we first summarize the current status and progress achieved in single-drug delivery strategies for the treatment of AC diseases. Subsequently, we focus mainly on advances in multiple drug delivery applications, including sequential release formulations targeting various pathological processes, synergistic targeting of the same pathological process, the spatial distribution in multiple tissues, and heterogeneous regeneration. We hope that this review will inspire the rational design of intra-articular drug delivery systems (DDSs) in the future.

Objective:To compare the effect of luteal-phase support administration of gonadotrophin-releasing hormone agonist(GnRH-a)on the outcomes of fresh embryo transfer in GnRH antagonist cycles,and explore a more appropriate protocol of luteal-phase support.Methods:A retrospective analysis was performed on the infer-tility patients who receiving in vitro fertilization or intracytoplasmic sperm injection embryo transfer(IVF/ICSI-ET)in the Reproductive Medicine Center of our hospital from January 2018 to December 2021.Our study collected clini-cal data from 674 cycles of infertility patients undergoing fresh cycle transplantation using antagonist regimens.The patients were divided into the control group(n =348)and the observation group(n =326)according to if Gn-RH-a was added to the luteal phase.The patients in control group were given standard luteal support treatment,while the patients in observation group were given multiple-dose GnRH-a 0.1mg to standard luteal support treat-ment after egg retrieval.The general data,ovulation induction and clinical outcome were compared between the two groups.Results:Between the two groups of patients,there was no statistically significant difference in age,years of infertility,body mass index(BMI),basal follicle stimulating hormone(bFSH),days and total amount of gonadotropin(Gn),chorionic gonadotropin(HCG),luteinizing hormone(LH),estradiol(E2)and progesterone(P),the number of retrieved oocytes,MII oocytes,number of embryos,normal fertilization rate,moderate to se-vere ovarian hyperstimulation syndrome(OHSS)rate,abortion rate,live birth rate,multiple pregnancy rate,inci-dence of pregnancy complications,birth weight of offspring,preterm birth rate,and birth defect rate(P>0.05).The implantation rate and clinical pregnancy rate were significantly higher in the observation group compared with control group(P<0.05).Multivariate Logistic regression analysis indicated that age and number of transferred embryos could influence the clinical pregnancy rate(OR 0.958,95%CI 0.917-1.000;OR 1.857,95%CI 1.173-2.942).Conclusions:In fresh embryo transfer cycles with GnRH antagonist protocol,on the basis of conventional luteal support,multi-dose intermittent short-acting GnRH-a for luteal support can achieve a good clin-ical outcome,and may be a suitable luteal support scheme for GnRH antagonist protocol in fresh embryo transfer cycles.

Objective:To investigate the impact of adding human menopausal gonadotropin (hMG) for in vitro fertilization-embryo transfer pregnancy outcomes in a standard population of non-advanced age with normal ovarian reserve function using a long follicular phase protocol.Methods:Clinical data of 489 patients with normal ovarian reserve function, who were admitted from January 2018 to January 2020 in the Affiliated Hospital of Guizhou Medical University and underwent in vitro fertilization for the first time with the long follicular phase protocol in fresh cycles, were retrospectively analyzed. The patients were divided into three groups according to whether or not to add urine-derived hMG and the timing of addition: non-addition group (group A), medium-term hMG group (group B1), whole course hMG group (group B2); the laboratory parameters of each group were observed, and the effect of ovulation induction drugs and pregnancy outcomes were compared.Results:The ages of B1 and B2 groups were significantly higher than that of group A ( P=0.019 and P=0.011). The basal FSH level of group B2 was significantly higher than those of group A and group B1 ( P<0.01 and P=0.006), and the basal FSH/LH ratio of group B2 was significantly higher than that of group B1 ( P=0.009). Antral follicle counts of group A and group B1 were significantly higher than that of group B2 ( P=0.007 and P=0.017). The superior embryo rate of group B2 [(47±27)%] was significantly higher than that of group A ( P=0.017). The embryo implantation rate of group B1 was significantly lower than those of group A and group B2 ( P=0.043 and P<0.01). The clinical pregnancy rate of group B2 [76.7% (155/202)] was significantly higher than those of group A ( P=0.039) and group B1 ( P<0.01). The live-birth rate of group B2 [67.3% (136/202)] was significantly higher than those of group A ( P=0.017) and group B1 ( P=0.001). Conclusions:For non-advanced aged patients with normal ovarian reserve function, the long protocol of follicular phase is suitable for those with relatively low ovarian reserve function. Adding hMG in the whole course of ovulation induction after gonadotropin-releasing hormone agonist reduction could improve the pregnancy outcomes by improving the quality of embryos.

Due to good mechanical properties and biocompatibility, tissue engineering scaffolds have become the vital method for repairing and regenerating articular cartilage defects. With the continuous development of tissue engineering technology, many scaffolds preparation and formation methods have been developed and tested in the past decade, however, the preparation of ideal regenerative scaffolds remain controversial. As load-bearing tissue inside the body joints, the matrix structure and cell composition of articular cartilage are hierarchical, and there are several smooth natural gradients from the cartilage surface to the subchondral bone layer, including cell phenotype and number, specific growth factors, matrix composition, fiber arrangement, mechanical properties, nutrient and oxygen consumption. Therefore, in the design of regenerative scaffolds, it is necessary to achieve these gradients to regenerate articular cartilage in situ. In recent studies, many new biomimetic gradient scaffolds have been used to simulate the natural gradient of articular cartilage. These scaffolds show different mechanical, physicochemical or biological gradients in the structure, and have achieved good repair effects. The related articles on tissue engineering for the treatment of articular cartilage defects were retrieved by searching databases with key wordsarticular cartilage injury, cartilage repair and gradient scaffolds. In this work,the structural, biochemical, biomechanical and nutrient metabolism gradients of natural articular cartilage were studied and summarized firstly. Then, the latest design and construction of articular cartilage gradient scaffolds were classified. Besides that, the material composition (such as hydrogels, nanomaterials, etc.) and the preparation process (such as electrospinning, 3D printing, etc.) of grandient scaffolds were further enhanced. Finally, the prospect and challenge of biomimetic gradient scaffolds in cartilage engineering are discussed, which provides a theoretical basis for the successful application of gradient scaffolds in clinical transformation.

Objective:To analyze the expression pattern, mechanism and clinical significance of melanoma-associated antigen-C2 (MAGE-C2) in tumor-free breast specimens, breast benign disease specimens and breast cancer specimens.Methods:Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to investigate the expressions of MAGE-C2 in 60 tumor-free breast specimens, 60 breast benign disease specimens and 60 breast cancer specimens. The correlation of MAGE-C2 expression with clinicopathological parameters and prognosis of breast cancer patients were analyzed. The expression of MAGE-C2 was also detected by RT-PCR in breast cancer cell MCF-7 and MDA-MB-231 treated with DNA methylase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA).Results:The positive expression rates of MAGE-C2 mRNA and protein were 61.7% (37/60) and 58.3% (35/60) in breast cancer specimens, respectively, while negative expressed in breast and begin disease specimens. MAGE-C2 protein expression was associated with tumor grade, histological type and blood vessel invasion of breast cancer patients ( P<0.05). The incidence of recurrence-free survival of patients with positive MAGE-C2 expression were lower than that of patients with negative MAGE-C2 expression ( P<0.05). Multivariate Cox regression analysis showed that the clinical stage ( P<0.01), lymph node metastasis ( P<0.05) and MAGE-C2 expression ( P<0.05) were the independent prognostic factors of breast cancer patients. The MAGE-C2 mRNA was not observed in the control and TSA treated breast cancer cells while upregulated in the 5-aza-CdR treated cells. Besides, 5-aza-CdR combined with TSA further enhanced MAGE-C2 mRNA level in breast cancer cells ( P<0.05). Conclusions:MAGE-C2 is one of the tumor-specific antigen and its expression is related with the poor prognosis of breast cancer patients. DNA methylation and histone acetylation may be an important regulation mechanism of MAGE-C2 gene expression.

Objective:To analyze the expression pattern, mechanism and clinical significance of melanoma-associated antigen-C2 (MAGE-C2) in tumor-free breast specimens, breast benign disease specimens and breast cancer specimens.Methods:Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to investigate the expressions of MAGE-C2 in 60 tumor-free breast specimens, 60 breast benign disease specimens and 60 breast cancer specimens. The correlation of MAGE-C2 expression with clinicopathological parameters and prognosis of breast cancer patients were analyzed. The expression of MAGE-C2 was also detected by RT-PCR in breast cancer cell MCF-7 and MDA-MB-231 treated with DNA methylase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA).Results:The positive expression rates of MAGE-C2 mRNA and protein were 61.7% (37/60) and 58.3% (35/60) in breast cancer specimens, respectively, while negative expressed in breast and begin disease specimens. MAGE-C2 protein expression was associated with tumor grade, histological type and blood vessel invasion of breast cancer patients ( P<0.05). The incidence of recurrence-free survival of patients with positive MAGE-C2 expression were lower than that of patients with negative MAGE-C2 expression ( P<0.05). Multivariate Cox regression analysis showed that the clinical stage ( P<0.01), lymph node metastasis ( P<0.05) and MAGE-C2 expression ( P<0.05) were the independent prognostic factors of breast cancer patients. The MAGE-C2 mRNA was not observed in the control and TSA treated breast cancer cells while upregulated in the 5-aza-CdR treated cells. Besides, 5-aza-CdR combined with TSA further enhanced MAGE-C2 mRNA level in breast cancer cells ( P<0.05). Conclusions:MAGE-C2 is one of the tumor-specific antigen and its expression is related with the poor prognosis of breast cancer patients. DNA methylation and histone acetylation may be an important regulation mechanism of MAGE-C2 gene expression.

[Abstract] Objective: To investigate the expression of MAGE-C1 (melanoma-associated antigen-C1) in breast cancer tissues and its correlation with clinicopathological features and prognosis of breast cancer patients. Methods: Breast cancer tissues, normal breast tissues and benign breast lesion tissues (60 samples for each) were collected from the Fourth Hospital of Hebei Medical University during January 2008 and December 2008.The mRNA and protein expressions of MAGE-C1 in three types of breast tissues were detected by RT-PCR and immunohistochemistry, and their correlation with clinicopathological parameters and prognosis of breast cancer patients were also analyzed. DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA) were used to treat breast cancer MDA-MB-231 and MCF-7 cells, and RT-PCR was used to determine the changes in mRNA expression of MAGE-C1 after drug treatment. Results: The positive expression rate of MAGE-C1 mRNA and protein in breast cancer tissues were 43.3% (26/60) and 38.3% (23/60), respectively; and the mRNA and protein expressions of MAGE-C1 were all negative in normal breast tissues and benign breast lesion tissues. MAGE-C1 expression was positively associated with high tumor grade (χ2 =6.233, P<0.05). Recurrence-free survival (RFS) of patients with negative MAGE-C1 expression was significantly longer than those patients with positive MAGE-C1 expression (χ 2 =4.213, P<0.05). MAGE-C1 expression (HR=3.980, P<0.05) and clinical stage (HR=3.637, P<0.05) could be used as independent prognostic factors for breast cancer patients. 5-Aza-CdR and/or TSA treatment had no significant influence on MAGE-C1 gene expression (P>0.05). Conclusion: MAGE-C1 is a tumor-specific antigen and its expression is associated with poor prognosis of breast cancer patients.

The treatment of articular cartilage (AC) injury caused by various reasons is still a major clinical problem. The emergence of cartilage tissue engineering brings new hope for the treatment of AC injury. In general, AC tissue engineering can be divided into two categories, including cell-based tissue engineering and cell-free tissue engineering. Although cell-based tissue engineering can repair cartilage damage to a certain extent, existing therapeutic strategies still suffer from limited cell sources, high costs, risk of disease transmission, and complex procedures. However, the cell-free tissue engineering avoids these shortcomings and brings hope for in-situ AC regeneration. Non-cellular tissue engineering is mainly used to recruit endogenous stem cells/progenitor cells (SCPCs) to reach the site of cartilage injury, and provide a suitable regenerative microenvironment to promote cell proliferation and chondrogenic differentiation, then the maturation of new cartilage tissue was promoted. Therefore, it is also called as cell-homing in situ tissue engineering. Successful recruitment of endogenous SCPCs is the first step in in-situ cartilage tissue engineering. This review aims to introduce chemokine response of cartilage injury, systematically summarize traditional chemoattractant (chemokines and growth factors etc.) and emerging chemoattractant (functional peptides, exosomes and nucleic acid adapters etc.), evaluate the combination mode between chemoattractant and delivery devices, discuss the prospects and challenges of chemoattractant-mediated in situ tissue engineering and provide theoretical basis for the design of endogenous SCPCs homing-based in situ tissue engineering.

Objective To investigate the clinical value of aspirin before pregnancy combined with low molecular heparin during pregnancy on treatment of patients with recurrent spontaneous abortion caused by prethrombotic state.Methods A total 60 patients with recurrent spontaneous abortion were randomly divided into treatment group and control group,30 cases in each group.The treatment group was treated with small dose of aspirin before pregnancy and low dosage molecular heparin during pregnancy,while the control group was treated with low dosage molecular heparin during pregnancy.Pregnancy outcome was compared between two groups.Results The partus maturus rate of treatment group was significantly higher than control group (P ＜ 0.05),and abortion rate was significantly lower than control group (P ＜ 0.05).Conclusion Aspirin before pregnancy combined with low molecular heparin during pregnancy is effective in treatment of patients with recurrent spontaneous abortion caused by prethrombotic state,which can increase partus maturus rate and reduce abortion rate.