Lymphatic metastasis is the main metastatic route for colorectal cancer, which increases the risk of cancer recurrence and distant metastasis. The properties of the lymph node metastatic colorectal cancer (LNM-CRC) cells are poorly understood, and effective therapies are still lacking. Here, we found that hypoxia-induced fibroblast activation protein alpha (FAPα) expression in LNM-CRC cells. Gain- or loss-function experiments demonstrated that FAPα enhanced tumor cell migration, invasion, epithelial-mesenchymal transition, stemness, and lymphangiogenesis via activation of the STAT3 pathway. In addition, FAPα in tumor cells induced extracellular matrix remodeling and established an immunosuppressive environment via recruiting regulatory T cells, to promote colorectal cancer lymph node metastasis (CRCLNM). Z-GP-DAVLBH, a FAPα-activated prodrug, inhibited CRCLNM by targeting FAPα-positive LNM-CRC cells. Our study highlights the role of FAPα in tumor cells in CRCLNM and provides a potential therapeutic target and promising strategy for CRCLNM.

Objective:To analyze the genetic and clinical characteristics, treatment and prognosis of patients diagnosed with maturity onset of diabetes of the young (MODY) 12 subtype.Methods:This retrospective study collected and analyzed data from 5 children with MODY12 subtype caused by ABCC8 gene variants who underwent inpatient and outpatient genetic testing at Beijing Children′s Hospital from January 2016 to December 2023. Their clinical and genetic features, treatment, and follow-up results were analyzed.Results:Among the 5 patients with MODY12 subtype, 4 were male and 1 was female, with an age of 13.4 (5.5, 14.6) years. Four of the patients were born large for gestational age, while one was born small for gestational age. Two patients were overweight or obese. Three patients exhibited typical symptoms of diabetes, while 2 were incidentally found to have elevated blood glucose level. One patient was found to have diabetic ketoacidosis at onset, who was diagnosed with congenital hyperinsulinism during the neonatal period and received diazoxide treatment, and experienced intellectual developmental delay. All 5 patients had autosomal dominant inherited diabetes within 3 generations. The fasting blood glucose at onset was 7.5 (6.5, 10.0) mmol/L, the haemoglobin A1c (HbA1c) was 11.8% (7.5%, 13.5%), and the fasting C-peptide was 1.2 (1.1, 2.2) μg/L. The duration of follow-up was 15 (9, 32) months. One patient underwent lifestyle intervention, 2 received metformin orally, 1 received insulin therapy, and the other received subcutaneous injection of insulin combined with sulfonylurea orally. At the last follow-up, the median fasting blood glucose was 6.1 (5.1, 7.0) mmol/L, the HbA1c was 5.9% (5.7%, 7.1%), and the fasting C-peptide was 1.7 (0.9, 2.9) μg/L. One patient developed diabetic retinopathy. There were 4 missense variations in ABCC8 gene and one in-frame deletion, all of which were maternally inherited heterozygotes.Conclusions:MODY12 subtype is a heterogeneous disorder with the age of onset from infancy to adolescence. It can present as mild hyperglycemia or diabetic ketoacidosis, and has a high incidence of obesity. Definitive diagnosis can be achieved through genetic test, and individualized treatment is recommended based on glucose levels.

Objective To investigate the acute toxicity,irritation,and other effects of Shexiang Anhe hemorrhoid ointment on hemorrhoid-phased model rats by examining the TLR4/p38 MAPK/NF-κB signaling pathway.Methods Sixteen New Zealand rabbits were randomly divided into an intact skin Shexiang Anhe hemorrhoid ointment group,intact skin control group,broken skin Shexiang Anhe hemorrhoid ointment group,and broken skin control group,with four rabbits in each group.In the experimental group,20 g of Shexiang Anhe hemorrhoid ointment(1 g/mL)was evenly applied to an area on the rabbits depleted of hair,and an equal volume of solvent(mixture of glycerol,lanolin,and water)was evenly applied to the area on the backs of the rabbits in the control group once a day for 14 days.Another 40 rats were taken and randomly divided into a normal group,model group,Maillard group,and hemorrhoid cream group,with 10 animals in each group.The cream was applied once a day for 14 days.The acute toxicity of the cream in the intact and broken skin of rabbits was observed by hematoxylin and eosin staining and other method after treatment.In situ photographs were taken of the perianal tissues of rats with hemorrhoids to observe the efficacy of Shexiang Anhe hemorrhoid ointment,and the length and width of the ulcers were measured with vernier calipers to calculate the area.Quantitative real time polymerase chain reaction was used to detect the expression of TLR4,p38 MAPK,and NF-κB mRNA in the perianal tissues of the rats.Results Compared with rabbits in the control groups with intact or broken skin,rabbits in the administered group showed no significant difference in body mass.The mean values for the irritation evaluation points for Shexiang Anhe hemorrhoid ointment on rabbits with broken skin for 1 h,24 h,48 h,and 72 h were 1.5,1,0.5,and 0.25,respectively,showing there was no obvious skin irritation.In the hemorrhoidal rats,Shexiang Anhe hemorrhoid ointment treatment significantly reduced hemorrhoid symptoms after 14 days administration;the ulcer area was significantly smaller(P＜0.05)and TLR4,p38 MAPK,and NF-κB mRNA levels were significantly lower compared with the findings in the model group(P＜0.05).Conclusions Shexiang Anhe hemorrhoid ointment is a safe topical treatment with minimal acute toxicity and irritation to the skin and achieved good efficacy in the treatment of hemorrhoidal rats.Its mechanism of action may be related to the inhibition of the TLR4/p38 MAPK/NF-κB signaling pathway.

ObjectiveTo investigate the effect and mechanism of Nongsuo Dangguiwan in improving ovarian oxidative stress in rats with ovarian dysfunction. MethodThirty-six adult female SD rats were randomly divided into normal group， model group， positive drug group （Femoston， 0.3 mg·kg^-1）， and high-， medium-， and low-dose groups of concentrated Nongsuo Dangguiwan （2.08， 4.16， 8.32 g·kg^-1）， with six rats in each group. Rats， except for those in the normal group， were injected with 80 mg·kg^-1 vinyl cyclohexene dioxide （VCD） per day for 14 consecutive days to induce ovarian dysfunction. From the 15th day， rats were treated with corresponding drugs by gavage， while those in the model group received 2 mL·kg^-1 saline， once daily for 28 consecutive days. The ovarian index， levels of related hormones including estradiol （E₂）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and anti-mullerian hormone （AMH） in serum， as well as superoxide dismutase （SOD） activity and glutathione （GSH） content in serum were measured by enzyme-linked immunosorbent assay （ELISA）. The malondialdehyde （MDA） content in serum was detected by the thiobarbituric acid （TAB） method. Ovarian morphology was observed by hematoxylin-eosin （HE） staining. The expression of nuclear factor E₂-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1）， SOD2， and SOD1 in ovarian tissues was detected by immunohistochemistry （IHC） and Western blot. ResultCompared with the normal group， the model group showed a significant reduction in growing follicles in the ovary， loose arrangement of granulosa cells in the follicle， decreased body weight， ovarian index， and serum AMH and E₂ levels， increased LH and FSH levels （P<0.01）， reduced levels of SOD and GSH in serum （P<0.01）， and increased MDA level （P<0.01）. Compared with the model group， the groups with drug intervention showed increased ovarian index （P<0.05， P<0.01）， increased serum E₂ level （P<0.05， P<0.01）， decreased FSH， AMH， and LH levels （P<0.05， P<0.01）， increased number of growing follicles in the ovary， potentiated SOD activity in serum， increased GSH content， decreased MDA content （P<0.05， P<0.01）， and up-regulated expression levels of Nrf2， HO-1， SOD2， and SOD1 proteins in ovarian tissues （P<0.05， P<0.01）. ConclusionNongsuo Dangguiwan can regulate serum hormone levels， increase the expression of Nrf2， HO-1， SOD2， and SOD1 in ovarian tissues， and improve ovarian antioxidant capacity to resist oxidative stress injury， thereby improving ovarian reserve function.

Objective:To determine the expression of Brahma-related gene 1 (BRG1) in cutaneous squamous cell carcinoma (cSCC) tissues and cells, and to investigate molecular mechanisms underlying the regulatory effect of its interaction with activating transcription factor 2 (ATF2) on the proliferation, migration and invasion of cSCC cells.Methods:From 2015 to 2021, 66 paraffin-embedded actinic keratosis (AK) tissue samples and 80 paraffin-embedded cSCC (including squamous cell carcinoma in situ) tissue samples were collected from the Department of Dermatology, Affiliated Hospital 2 of Nantong University, and the diagnoses of all the cases were confirmed histopathologically; at the same time, 35 paraffin-embedded normal skin tissue samples obtained by cosmetic surgery served as normal control group. Immunohistochemical staining was performed to determine the BRG1 expression in cSCC, AK, and normal skin tissues, and correlations between BRG1 expression and clinicopathological parameters of cSCC patients were analyzed. Fresh tissue samples were collected from 12 cSCC patients and 12 healthy controls, and cSCC cell lines A431 and Scl-1 and a human immortalized keratinocyte cell line HaCaT were routinely cultured; real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to determine the mRNA expression of BRG1 in tissues and cells, and co-immunoprecipitation assay and cellular immunofluorescence staining were conducted to analyze the interaction between BRG1 and ATF2. The expression of BRG1 (BRG1 siRNA1 - 5 groups) and ATF2 (ATF2-shRNA group) in A431 and Scl-1 cells was knocked down by RNA interference, and cells transfected with negative control siRNA or shNC served as controls (control siRNA group and shNC group, respectively), cell counting kit-8 (CCK8) assay, colony formation assay, cell scratch assay, and Transwell assay were conducted to evaluate effects of knocking down BRG1 and ATF2 on the proliferation, migration, and invasion of cSCC cells. Comparisons of measurement data among multiple groups were conducted using one-way analysis of variance, and multiple comparisons were conducted using Dunnett- t test. Results:Immunohistochemical staining showed that the expression intensity of BRG1 protein was significantly lower in the cSCC and AK tissues than in the normal skin tissues ( χ2 = 44.40, P < 0.001). qRT-PCR showed that the mRNA expression level of BRG1 was significantly lower in the cSCC tissues (1.345 ± 0.956) than in the normal skin tissues (2.499 ± 1.501, t = 2.25, P = 0.035), and also significantly lower in A431 and Scl-1 cells (0.041 ± 0.002, 0.026 ± 0.003, respectively) than in HaCaT cells (0.135 ± 0.033, t = 4.95, 5.73, P = 0.008, 0.005, respectively). The low expression of BRG1 was associated with tumors at sun-exposed sites ( P = 0.041), low tumor differentiation ( P = 0.001), and high Broder′s grade ( P < 0.001) in the cSCC patients. In both A431 cells and Scl-1 cells, the BRG1 siRNA1 group and BRG1 siRNA2 group showed significantly increased numbers of cell colonies, migratory cells and invasive cells, as well as cell migration rates compared with the control siRNA group (all P < 0.05). Co-immunoprecipitation assay showed that BRG1 protein could bind to ATF2 protein in A431 and Scl-1 cells, and immunofluorescence staining showed that the two proteins were co-localized; compared with the control siRNA group, the BRG1 siRNA1 group (both A431 and Scl-1 cells) and BRG1 siRNA2 group (A431 cells) both showed increased phosphorylation and activation of ATF2 (all P < 0.05) ; in both A431 cells and Scl-1 cells, the shATF2 group showed significantly decreased numbers of cell colonies (both P = 0.001), cellular proliferative activity at 24 - 96 hours (all P < 0.001), and numbers of migratory cells and invasive cells compared with the shNC group (all P ≤ 0.001) . Conclusion:BRG1 was lowly expressed in the cSCC and AK tissues, and could inhibit the proliferation, migration, and invasion of cSCC cells; ATF2 could promote the proliferation, migration, and invasion of cSCC cells; BRG1 may exert an anti-tumor effect by interacting with ATF2 protein and inhibiting phosphorylation-dependent activation of ATF2.

ObjectiveTo explore the mechanism of Jianpi Yishen Huazhuo prescription in the improvement of ovarian function in polycystic ovary syndrome （PCOS）. MethodSeventy female SD rats in SPF grade were randomly divided into 6 groups， 15 in the blank group and 15 in the model group， 10 in the metformin group （0.1 g·kg^-1·d^-1）， and 10 in the low （1.275 g·kg^-1·d^-1）， medium （2.55 g·kg^-1·d^-1）， and high-dose （5.10 g·kg^-1·d^-1） Jianpi Yishen Huazhuo prescription groups. The blank group was given normal saline （10 mL·kg^-1·d^-1） by gavage and ordinary feed， and the other groups were given letrozole （1 mg·kg^-1·d^-1） by gavage combined with high-fat feed for 21 days to induce the model of PCOS. After modeling， the blank group and model group were given equal volume normal saline by gavage， and each drug group was given the corresponding dose of the drug by gavage for 30 days. The changes in body mass and fasting blood glucose （FPG） of rats before and after modeling were compared. Hematoxylin eosin （HE） staining was used to observe the morphological change in the ovaries of rats in each group. The serum levels of follicle stimulating hormone （FSH）， luteinizing hormone （LH）， testosterone （T）， anti-Mullerian hormone （AMH）， and estradiol （E₂） were measured by enzyme-linked immunosorbent assay （ELISA）， and the LH/FSH ratio was calculated. Immunohistochemical staining （IHC） and Western blot were used to detect the protein expression levels of nucleoside binding oligomerization domain protein like receptor 3 （NALP3）， apoptosis-associated speck-like protein （ASC）， cysteine protease-1 （Caspase-1）， nuclear transcription factor-κB （NF-κB）， interleukin-1β （IL-1β）， interleukin-18 （IL-18）， and interleukin-6 （IL-6） in the rat ovaries. ResultAs compared with the blank group， large follicles with polycystic expansion were found in the ovaries of the model group， no dominant follicles were found， the granular layer of follicles decreased and arranged loosely， and the number of corpus luteum decreased significantly. Serum T， LH， AMH and LH/FSH increased in the model group （P<0.05， P<0.01）， while FSH and E₂ decreased （P<0.05， P<0.01）. The relative protein expression levels of NALP3， ASC， Caspase-1， NF-κB， IL-1β， IL-18， and IL-6 increased （P<0.05， P<0.01） in the ovaries of the model group. Compared with the model group， the low， medium， and high-dose Jianpi Yishen Huazhuo prescription groups and the metformin group showed growing follicles and corpus luteum at all levels， the number of cystic expanding follicles decreased， the thickness of follicular granular layer increased， the number of follicular fluid increased， mature follicles were visible， and the local morphology of oocytes was complete. Serum T， LH， AMH， and LH/FSH in these groups decreased （P<0.05， P<0.01）， while E₂ and FSH increased （P<0.05）. The relative protein expressions of NALP3， ASC， Caspase-1， NF-κB， IL-1β， IL-18， and IL-6 in the ovaries of these groups decreased （P<0.05， P<0.01）. There was no significant difference among the treatment groups. ConclusionBy inhibiting the activation of NLRP3 inflammasome， Jianpi Yishen Huazhuo prescription reduces the release of NALP3， ASC， Caspase-1， NF-κB， IL-18， IL-1β， and IL-6 inflammatory factors in ovarian tissues， regulates endocrine level， and effectively reduces PCOS inflammatory statu， so as to play a role in improving ovarian function.

The p21 activated kinase 4 (PAK4) is serine/threonine protein kinase that is critical for cancer progression. Guided by X-ray crystallography and structure-based optimization, we report a novel subseries of C-3-substituted 6-ethynyl-1H-indole derivatives that display high potential and specificity towards group II PAKs. Among these inhibitors, compound 55 exhibited excellent inhibitory activity and kinase selectivity, displayed superior anti-migratory and anti-invasive properties against the lung cancer cell line A549 and the melanoma cell line B16. Compound 55 exhibited potent in vivo antitumor metastatic efficacy, with over 80% and 90% inhibition of lung metastasis in A549 or B16-BL6 lung metastasis models, respectively. Further mechanistic studies demonstrated that compound 55 mitigated TGF-β1-induced epithelial-mesenchymal transition (EMT).

Objective:To analyze the clinical features and mutation of myeloid differentiation factor 88 (MYD88) L265P in patients with diffuse large B-cell lymphoma (DLBCL) of central nervous system (CNS).Methods:The clinicopathological materials of 45 cases of DLBCL of CNS were retrospectively collected in Xuanwu Hospital, Capital Medical University from September 2014 to February 2017. The clinicopathological data were retrospectively analyzed, combined with immunohistochemistry, EB virus in situ hybridization, imaging and medical history. The mutation of MYD88 L265P gene was detected by pyrosequencing and its clinical significance was analyzed. Results:The age of the patients ranged from 42 to 82 years [(57.6±8.8) years], including 24 males and 21 females. Totally 93.3% (42/45) of the patients had supratentorial tumours, which were single or multiple. The cerebral hemisphere (31/45, 68.9%) was the most common involved site, and 21 cases (21/45, 46.7%) had multiple lesions. Histologically, DLBCL in the CNS showed diffuse infiltration of tumor tissue, some of which grew around blood vessels in a "sleeve" arrangement. CD 20 and CD 79a were diffusely and strongly positive. Thirty-nine cases (39/45, 86.7%) were non-germinal center B cell (non-GCB) subtype and 6 cases (6/45, 13.3%) were germinal center B cell (GCB) subtype. MYD88 L265P mutation was found in 64.4% (29/45) patients. There was statistically significant difference between non-GCB type (71.8%, 28/39) and GCB type DLBCL (1/6, P=0.017). Compared with the operation/biopsy group without chemotherapy, operation+chemotherapy, biopsy+chemotherapy, operation/biopsy+chemotherapy+stem cell transplantation can improve the survival and prognosis ( HR=0.05, 95% CI 0.01-0.33 , P=0.002; HR=0.04, 95% CI 0.01-0.36 , P=0.004; HR=0.01, 95% CI 0.00-0.17 , P=0.001; respectively). Conclusions:DLBCL of the CNS is aggressive tumor with poor prognosis, the clinical manifestations are complex and diverse, and the diagnosis is challenging. MYD88 L265P is a common and specific gene mutation in primary CNS lymphoma(PCNSL), which is of great significance in the diagnosis and treatment of lymphoma. The MYD88 L265P mutation was more frequently detected in non-GCB than GCB subtype. Chemotherapy can improve the survival rate of PCNSL patients. If chemotherapy achieves complete remission and autologous hematopoietic stem cell transplantation is performed, there may be a chance of long-term survival.

Objective:To explore the prognostic value of lymphocyte subsets in adult hemophagocytic syndrome (HPS).Methods:A total of 172 adult HPS patients diagnosed in 8 medical centers from January 2013 to August 2020 were selected for the study, of whom 87 were male (50.6%, 87/172), and 85 were female (49.4%, 85/172), with 68 survivors and 104 deaths. The clinical data were summarized, and variables such as lymphocyte subsets, immunoglobulin characteristics and fibrinogen were retrospectively analyzed, and the correlation between the mentioned variables and patient prognosis was analyzed. The optimal cut-off values of continuous variables were calculated by MaxStat, and the prognostic factors of HPS patients were screened based on the Cox proportional hazard regression model.Results:The median age of HPS patients was 56 (42, 66) years old, and the 5-year cumulative survival rate was 37.4% (37.4/100). The median age, platelet and albumin were 48 (27, 63) years, 84×10 9/L and 32.3 g/L in the survival group, and 59 years, 45.5×10 9/L, and 27.3 g/L in the death group, respectively. The differences between the two groups was statistically significant ( Z=?3.368, P=0.001; Z=?3.156, P=0.002; Z=?3.431, P=0.001). Patients with differentiated cluster 8+(CD8+)<11.1%, CD3+<64.9%, CD4+>51%, and CD4/CD8 ratio>2.18 had poor prognosis (χ 2=7.498, P=0.023; χ 2=4.169, P=0.041; χ 2=4.316, P=0.038; χ 2=9.372, P=0.002). Multivariable analysis showed that CD4/CD8 ratio, age, fibrinogen and hemoglobin were independent prognostic factors in HPS patients ( HR=2.435, P=0.027; HR=5.790, P<0.001; HR=0.432, P=0.018; HR=0.427, P=0.018). Conclusion:Peripheral blood lymphocyte subsets can be used to evaluate the prognosis of patients with HPS; CD4/CD8 ratio, age, fibrinogen, and hemoglobin are independent prognostic factors in HPS patients.

Objective:Factors influencing the prognosis of hemophagocytic lymphohistiocytosis (HLH) in adults were analyzed based on multicentric data.Methods:Clinical data of 124 adult patients with HLH diagnosed in eight medical centers in the Huaihai Lymphoma Working Group from March 2014 to July 2020 were collected. The optimal truncation value of continuous variables was obtained based on the Maxstat algorithm, X-Tile software, and restricted cubic spline. Cox proportional risk regression model was used to construct the adult HLH risk prediction model, and the visualization of the model was realized through the histogram. The bootstrap resampling method was used to verify the model, C-index and calibration curve was used to verify the histogram, and the prediction accuracy was checked. Kaplan-Meier analysis was used to calculate the survival rate and draw the survival curve. Furthermore, the differences between groups were tested by log-rank.Results:The median age of the 124 patients was 55 (18-84) years, including 61 (49.19%) males. The most common etiology was infection. Serum ferritin increased in 110 cases (88.71%) , hepatosplenomegaly in 57 cases (45.97%) . Of the 124 patients, 77 (62.10%) died, and the median survival time of the patients was 7.07 months. Univariate results showed that the prognosis of adult HLH was influenced by sex, age, fibrinogen, serum creatinine, alanine aminotransferase, and albumin ( P<0.05) . The results of multivariate analysis showed that gender, platelet, albumin, alanine aminotransferase, and treatment regimens were independent influencing factors for prognosis. Based on the above five risk factors, the prediction model of the histogram was established, and the C-index of the model was 0.739. Finally, the calibration chart showed good consistency between the observed and predicted values of HLH. Conclusion:The prognosis of the adult hemophagocytic syndrome is influenced by many factors. Gender, platelet, albumin, alanine aminotransferase, and treatment regimens are independent risk factors. Therefore, the established histogram provides a visual tool for clinicians to evaluate the prognosis of adult HLH.