[Mechanism of protective effect of resveratrol on poor ovarian response in mice].

This study aims to investigate the therapeutic effects and potential mechanisms of resveratrol(Res) on poor ovarian response(POR) in mice. The common target genes shared by Res and POR were predicted by network pharmacology, used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and then validated by animal experiments. The mice with regular estrous cycle after screening were randomized into normal, POR, and low-and high-dose(20 and 40 mg·kg~(-1), respectively) Res groups. The normal group was administrated with an equal volume of 0.9% sodium chloride solution by gavage, and the mice in other groups with tripterygium glycosides suspension(50 mg·kg~(-1)) by gavage for 2 weeks. After the modeling, the mice in low-and high-dose Res groups were treated with Res by gavage for 2 weeks, and the mice in normal and POR groups with an equal volume of 0.9% sodium chloride solution by gavage. Ovulation induction and sample collection were carried out on the day following the end of treatment. Vaginal smears were collected for observation of the changes in the estrous cycle, the counting of retrieved oocytes, and the measurement of ovarian wet weight and ovarian index. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of anti-mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in the serum. The ovarian tissue morphology and granulosa cell apoptosis were observed by hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL), respectively. Western blot was employed to determine the protein levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(AKT), forkhead box O(FOXO) 3a, hypoxia-inducible factor(HIF)-1α, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). A total of 222 common targets shared by Res and POR were collected. GO annotation indicated that these targets were mainly involved in oxidative stress response. KEGG enrichment analysis revealed that Res can intervene in POR via PI3K/AKT, HIF-1, and FOXO signaling pathways. Animal experiments showed that the model group had higher rate of estrous cycle disorders, lower number and poorer morphology of normally developed follicles at all levels, more atretic follicles, higher apoptosis of ovarian granulosa cells, lower number of retrieved oocytes, lower ovarian wet weight and ovarian index, higher serum levels of FSH and LH, lower levels of AMH and E_2, higher expression levels of HIF-1α, FOXO3a and Bax, and lower expression levels of PI3K, AKT, and Bcl-2 in the ovarian tissue than the normal group. Compared with the POR group, low-and high-dose Res decreased the rate of estrous cycle disorders, improved the follicle number and morphology, reduced atretic follicles, promoted the apoptosis of ovarian granulosa cells, increased retrieved oocytes, ovarian wet weight and ovarian index, and lowered serum FSH and LH levels. Moreover, Res down-regulated the expression levels of HIF-1α, FOXO3a and Bax, and up-regulated the expression levels of PI3K, AKT and Bcl-2 in the ovarian tissue. In summary, Res can inhibit apoptosis and mitigate poor ovarian response in mice by regulating the PI3K/AKT/FOXO3a and HIF-1α pathways.

Genome-wide analysis of the role of DNA methylation in inbreeding depression of reproduction in Langshan chicken.

Inbreeding depression of chicken reproduction is a major concern in the conservation of chicken genetic resources. To investigate the potential DNA methylation sites involved in the inbreeding depression of chicken reproduction, we carried out whole-genome bisulfite sequencing (WGBS) of hypothalamus and ovary tissues from the strongly and weakly inbred Langshan chickens, respectively. 5948 and 4593 differentially methylated regions (DMRs) were identified in the hypothalamus and ovary between the strongly and weakly inbred Langshan chickens, respectively. Large numbers of DMR-related genes (DMGs) were enriched in reproduction-related pathways. By combining the WGBS and transcriptome data, two DMRs in SRD5A1 and CDC27 genes were inferred as the most likely biomarkers of inbreeding depression of reproduction in Langshan chicken. Our study provides the first systematic investigation of the DNA methylation changes in strongly inbred chickens, and extends our understanding of the regulatory mechanisms underlying inbreeding depression in chicken reproduction.

Annonaceous acetogenin mimic AA005 suppresses human colon cancer cell growth in vivo through downregulation of Mcl-1.

Annonaceous acetogenins are a well-established family of natural products with significant bioactivities, especially high cytotoxic and antitumor activities. AA005 is an annonaceous acetogenin mimic that has shown significant cytotoxicity against a variety of cancer cell lines, but its in vivo antitumor effects have not been demonstrated so far, and its anticancer mechanisms remain ambiguous. In this study, we investigated the effects of AA005 on human colon cancer cell lines in vivo. Human colon carcinoma cell line SW620 xenograft nude mice were treated with AA005 (5 mg/kg/day, i.p.) for 21 days. AA005 administration markedly inhibited the tumor growth via promoting nuclear translocation of apoptosis-inducing factor (AIF) and inducing AIF-dependent cell death. Subsequent studies in human colon carcinoma cell lines SW620 and RKO in vitro revealed that after the colon cancer cells exposed to AA005, downregulation of a B-cell lymphoma 2 family protein, myeloid cell leukemia-1 (Mcl-1), was an early event due to the inhibition of Mcl-1 mRNA level and protein synthesis in a time-dependent manner. Intriguingly, knockdown of Mcl-1 using small interfering RNA markedly accelerated the nuclear translocation of AIF and upregulation of receptor interacting protein-1, and enhanced AA005-mediated lethality, whereas ectopic expression of Mcl-1 substantially attenuated AA005-mediated lethality in the colon cancer cells. Finally, silencing Mcl-1 expression markedly enhanced AA005-induced lethality in SW620 xenograft nude mice, demonstrating a pivotal role of Mcl-1 downregulation in mediating the in vivo antitumor effects of AA005. Taken together, this study demonstrates for the first time the anticancer effects of AA005 against human colon cancer cell lines in vivo, which is mediated through the downregulation of Mcl-1.

Type I alveolar epithelial cells mount innate immune responses during pneumococcal pneumonia.

Pneumonia results from bacteria in the alveoli. The alveolar epithelium consists of type II cells, which secrete surfactant and associated proteins, and type I cells, which constitute 95% of the surface area and meet anatomic and structural needs. Other than constitutively expressed surfactant proteins, it is unknown whether alveolar epithelial cells have distinct roles in innate immunity. Because innate immunity gene induction depends on NF-κB RelA (also known as p65) during pneumonia, we generated a murine model of RelA mutated throughout the alveolar epithelium. In response to LPS, only 2 of 84 cytokine transcripts (CCL20 and CXCL5) were blunted in lungs of mutants, suggesting that a very limited subset of immune mediators is selectively elaborated by the alveolar epithelium. Lung CCL20 induction required epithelial RelA regardless of stimulus, whereas lung CXCL5 expression depended on RelA after instillation of LPS but not pneumococcus. RelA knockdown in vitro suggested that CXCL5 induction required RelA in type II cells but not type I cells. Sorted cell populations from mouse lungs revealed that CXCL5 was induced during pneumonia in type I cells, which did not require RelA. TLR2 and STING were also induced in type I cells, with RelA essential for TLR2 but not STING. To our knowledge, these data are the first direct demonstration that type I cells, which constitute the majority of the alveolar surface, mount innate immune responses during bacterial infection. These are also, to our knowledge, the first evidence for entirely RelA-independent pathways of innate immunity gene induction in any cell during pneumonia.

[Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on PI3K/AKT/FOXO3a pathway in mice with poor ovarian response].

OBJECTIVE: To observe the protective effect of acupuncture at "Zhibian" (BL 54) through "Shuidao (ST 28)" based on the PI3K/AKT/FOXO3a pathway in mice with poor ovarian response (POR), and to explore the possible mechanism of acupuncture in inhibiting ovarian granulosa cells apoptosis in POR. METHODS: A total of 45 mice with regular estrous cycles were randomly divided into a blank group, a model group and an acupuncture group, with 15 mice in each group. Mice in the model group and the acupuncture group were given triptolide suspension (50 mg•kg-1•d-1) by gavage for 2 weeks to establish POR model. After successful modeling, mice in the acupuncture group were given acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) for 2 weeks, once a day, 20 min each time. Ovulation induction was started the day after the intervention ended, and samples were taken from each group after ovulation induction. Vaginal smears were used to observe changes in the estrous cycle of mice. The number of oocytes retrieved, ovarian wet weight, final body weight, and ovarian index were measured. The levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), estradiol (E2), and luteinizing hormone (LH) in serum were detected by ELISA. The morphology of ovarian tissue was observed by HE staining. The apoptosis of ovarian granulosa cells was detected by TUNEL staining. The mRNA expression of PI3K, AKT, and FOXO3a in ovarian tissue was detected by real-time fluorescence quantitative PCR. The protein expression of Bcl-2 associated X protein (BAX), caspase-3, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) in ovarian tissue was detected by Western blot. RESULTS: Compared with the blank group, the rate of estrous cycle disorder in the model group was increased (P<0.01); compared with the model group, the rate of estrous cycle disorder in the acupuncture group was decreased (P<0.01). Compared with the blank group, the number of oocytes retrieved, ovarian wet weight, ovarian index, and final body weight in the model group were decreased (P<0.01); compared with the model group, the number of oocytes retrieved, ovarian index, and ovarian wet weight were increased (P<0.01, P<0.05), and there was no significant difference in final body weight (P>0.05) in the acupuncture group. Compared with the blank group, the serum levels of FSH and LH were increased (P<0.01), and the serum levels of AMH and E2 were decreased (P<0.01) in the model group; compared with the model group, the serum levels of FSH and LH were decreased (P<0.01, P<0.05), and the serum levels of AMH and E2 were increased (P<0.01, P<0.05) in the acupuncture group. Compared with the blank group, the number of normal developing follicles in ovarian tissue in the model group was decreased and the morphology was poor, while the number of atretic follicles increased; compared with the model group, the number, morphology, and granulosa cell structure of follicles in the acupuncture group improved to varying degrees, and the number of atretic follicles decreased. Compared with the blank group, the apoptosis rate of ovarian granulosa cells in the model group was increased (P<0.01); compared with the model group, the apoptosis rate of ovarian granulosa cells in the acupuncture group was decreased (P<0.01). Compared with the blank group, the FOXO3a mRNA expression and caspase-3 and BAX protein expression in ovarian tissue in the model group were increased (P<0.01), and the mRNA expression of PI3K and AKT and the protein expression of p-PI3K, p-AKT, and p-FOXO3a in ovarian tissue were decreased (P<0.01); compared with the model group, the mRNA expression of FOXO3a and protein expression of caspase-3 and BAX in ovarian tissue in the acupuncture group were decreased (P<0.05, P<0.01), and the mRNA expression of PI3K and AKT and the protein expression of p-PI3K, p-AKT, and p-FOXO3a in ovarian tissue were increased (P<0.01, P<0.05). CONCLUSION: Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could inhibit ovarian cell apoptosis, and improve ovarian function in POR mice, and its mechanism may be related to the regulation of key factors in the PI3K/AKT/FOXO3a pathway.

Ellagic acid promotes osteoblasts differentiation via activating SMAD2/3 pathway and alleviates bone mass loss in OVX mice.

Characterized by bone mass loss, osteoporosis is an orthopedic disease typically found in postmenopausal women and aging individuals. Consistent with its pathogenesis summarized as an imbalance in bone formation/resorption, current pharmacologically therapeutic strategies for osteoporosis mainly aim to promote bone formation or/and inhibit bone resorption. However, few effective drugs with mild clinical side effects have been developed, making it a well-concerned issue to seek appropriate drugs for osteoporosis. In this study, we investigated the effect of ellagic acid (EA) on osteogenesis in vitro and in vivo and searched for its molecular mechanism. Here, we showed that EA promoted osteogenic differentiation of MSCs, increased mRNA and protein expression levels of osteoblast marker genes Runt-related transcription factor2, Osterix, Alkaline phosphatase, Collagen type I alpha 1, Osteopontin and Osteocalcin. Furthermore, ovariectomized mice with orally administered EA (10 mg/kg, 50 mg/kg) had significantly higher bone mass than those in controls. And experiments such as fluorescence double-labeling and enzyme-linked immunosorbent assay also demonstrated that EA could promote osteogenesis in vivo. To probe the molecular mechanism of EA, we performed RNA sequencing analysis using EA-treated BMSCs. Significant up-regulation of SMAD2/3 transcription factors was identified by RNA-seq, and it was confirmed in vitro that EA promoted bone formation by activating the SMAD2/3 signaling pathway. Evidence from our present experiments indicates that EA may be a promising candidate for clinical treatment for osteoporosis in future.

Changes in PI3K/AKT and NRF2/HO-1 signaling expression and intestinal microbiota in bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is the outcome of the prolonged interstitial pneumonia, characterized by excessive accumulation of fibroblasts and collagen deposition, leading to its development. This study aimed to study the changes in PI3K/AKT and NRF2/HO-1 signaling expression and intestinal microbiota in a rat model of a novel bleomycin-induced pulmonary fibrosis. The findings of our study showed the model was successfully established. The results showed that the alveolar septum in the model was significantly widened and infiltrated by severe inflammatory cells. Alveolar atrophy occurred due to the formation of multiple inflammatory foci. During this period, fibrous tissue was distributed in strips and patches, primarily around the pulmonary interstitium and bronchus. Moreover, lung damage and fibrosis progressively worsened over time. The mRNA expression of HO-1 and NRF2 in the model decreased while the mRNA expression of HIF-1α, VEGF, PI3K and AKT increased. Furthermore, it was observed to decrease the protein expression of E-cad, HO-1 and NRF2, and increase the protein expression of α-SMA and p-AKT. Additionally, this model leaded to an imbalance in the intestinal microbiota. This study demonstrate that the novel pulmonary fibrosis model activates the NRF2/HO-1 pathway and the PI3K/AKT pathway in rat lung tissues, and leading to intestinal barrier disorder.

Origin, development and advantageous disease spectrum of the "Zhibian (BL 54) through Shuidao (ST 28)" needling technique. / "秩边透水道"é’ˆæ³•æºæµæŽ¢æžåŠä¼˜åŠ¿ç— è°±.

This paper systematically reviews the historical evolution of the "Zhibian (BL 54) through Shuidao (ST 28)" needling technique by examining the history of the Mang needle, controversies and positioning of the Zhibian (BL 54), and the formation and essentials of the needling technique. Furthermore, the advantageous disease spectrum of this needling technique is summarized, and speculates on potential advantageous disease spectrum from the neural mechanisms of obtaining qi and achieving efficacy. Lastly, this paper discusses the inadequacies in the research on " Zhibian (BL 54) through Shuidao (ST 28)" needling technique, aiming to provide a comprehensive understanding and reference for further research on this technique.

Development and validation of bioanalytical assays for the quantification of 9MW2821, a nectin-4-targeting antibody-drug conjugate.

We designed and developed 9MW2821, an anti-Nectin-4 antibody-drug conjugate (ADC) with an enzymatically cleavable valine-citrulline linker and monomethyl auristatin E (MMAE) as the payload. Four bioanalytical assays for total antibodies, conjugated antibodies, conjugated payload, and free payload were then developed and validated for the comprehensive evaluation of the multiple drug forms of 9MW2821. Specific sandwich enzyme-linked immunosorbent assays were used to quantify total antibodies and conjugated antibody, showing good drug-to-antibody ratio (DAR) tolerance. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine free MMAE, and conjugated MMAE was quantified using a combination of ligand-binding assay (LBA) and LC-MS/MS. Based on these four assays, we studied the serum stability and monkey pharmacokinetic profiles of 9MW2821, and the in vivo DAR of 9MW2821 was calculated and dynamically monitored. In conclusion, we developed and validated series of bioanalytical assays to quantify multiple forms of 9MW2821, a new ADC, and used the assays to evaluate the serum stability and monkey pharmacokinetic characteristics. The results indicate good linker stability and suggest that the developed assays can be further used in clinical settings.

The transcription factors Grainyhead-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differentiation.

The Grainyhead family of transcription factors controls morphogenesis and differentiation of epithelial cell layers in multicellular organisms by regulating cell junction- and proliferation-related genes. Grainyhead-like 2 (Grhl2) is expressed in developing mouse lung epithelium and is required for normal lung organogenesis. The specific epithelial cells expressing Grhl2 and the genes regulated by Grhl2 in normal lungs are mostly unknown. In these studies we identified the NK2-homeobox 1 transcription factor (Nkx2-1) as a direct transcriptional target of Grhl2. By binding and transcriptional assays and by confocal microscopy we showed that these two transcription factors form a positive feedback loop in vivo and in cell lines and are co-expressed in lung bronchiolar and alveolar type II cells. The morphological changes observed in flattening lung alveolar type II cells in culture are associated with down-regulation of Grhl2 and Nkx2-1. Reduction of Grhl2 in lung epithelial cell lines results in lower expression levels of Nkx2-1 and of known Grhl2 target genes. By microarray analysis we identified that in addition to Cadherin1 and Claudin4, Grhl2 regulates other cell interaction genes such as semaphorins and their receptors, which also play a functional role in developing lung epithelium. Impaired collective cell migration observed in Grhl2 knockdown cell monolayers is associated with reduced expression of these genes and may contribute to the altered epithelial phenotype reported in Grhl2 mutant mice. Thus, Grhl2 functions at the nexus of a novel regulatory network, connecting lung epithelial cell identity, migration, and cell-cell interactions.

Electroacupuncture for ulcerative colitis patients: A meta-analysis and acupoints selection study.

Objective: To evaluate the efficacy and safety of electroacupuncture (EA) on ulcerative colitis (UC) and explore the influence of EA parameters and acupoint compatibility to optimize the clinical treatment plan. Methods: After searching eight databases, data were extracted and analyzed to determine the quality and bias of the study's methodological design, and randomized controlled trial (RCT) datas were meta-analyzed. Results: Twelve studies that meet the criteria were included. The results of meta-analysis indicated that, compared with the control group, experimental group had better clinical efficacy [RR = 1.27, 95%CI = (1.19, 1.36), P < 0.01], Other indicators such as cure rate [RR = 1.73, 95%CI = (1.43, 2.09), P < 0.01], effective rate of mucosal lesions under enteroscopy [RR = 1.24, 95%CI = (1.11, 1.38), P < 0.01], serum inflammatory factor TNF-α [MD = -41.11, 95%CI = (-46.01, 36.22), P < 0.01] were significantly better than those in the control group. Sixteen acupoints on the Ren, Bladder, Stomach, Spleen, and Liver meridians were used 74 times. RN4-ST25 is the most compatible acupoints. Conclusion: The clinical efficacy of EA in treating UC is superior than the control group's, and it has curative effects in terms of cure rate, efficacy of mucosal lesions under colonoscopy, serum inflammatory factors, and Traditional Chinese Medicine (TCM) syndrome scores. Combining acupoints of the Bladder, Stomach, and Ren meridians and using dense wave for 30 min each time for more than 6 weeks may be optimal for UC patients.

Study on the mechanism of electroacupuncture regulating macrophage polarization to improve ulcerative colitis in rats. / ç”µé’ˆè°ƒèŠ‚å·¨å™¬ç»†èƒžæžåŒ–æ”¹å–„å¤§é¼ æºƒç–¡æ€§ç»“è‚ ç‚Žçš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To investigate the effect of electroacupuncture(EA) at "Changbing Decoction" on alleviating ulcerative colitis (UC) and regulating the polarization of colonic macrophages in rats, so as to explore its mechanisms underlying improvement of UC. METHODS: Twenty-six male SD rats were randomly divided into 4 groups：normal group(6 rats), model group(8 rats), EA group(6 rats), and western medication group(6 rats). The rat model of UC was established by using 5% dextran sulfate sodium (DSS) solution drinking water for 7 days, followed by drinking 1% DSS solution during treatment period. After 7-day model establishment, EA treatment(10 Hz/50 Hz, 20 min) was applied to "Zhongwan"(CV12), bilateral "Tianshu"(ST25) and "Shangjuxu"(ST37) for 3 d, and rats in the western medication group were given mesalazine suspension(200 mg/kg) by gavage for 3 d. The body weight, spleen weight and colon length of rats were measured. The disease activity index (DAI) score was evaluated. The morphological changes and inflammatory cell infiltration of colon were detected after HE staining and pathological scores were eva-luated. The contents of tumor necrosis factor α(TNF-α), interleukin(IL)-1ß, IL-2 and IL-10 in serum were detected by ELISA. The protein expressions of M1 and M2 macrophage markers nitric oxide synthase (iNOS) and arginase 1(Arg1) were detected by fluorescence double staining and Western blot, respectively. Quantitative real-time PCR was used to detect iNOS and Arg1 mRNA expressions. RESULTS: Compared with the normal group, rats in the model group had increased pathological damage degree and inflammatory cell infiltration in the colon tissue, slowed-down body weight gain, decreased colon length, spleen weight, serum anti-inflammatory factors IL-2 and IL-10 contents, colonic Arg1/CD68 fluorescence positive expression, and Arg1 protein and mRNA expressions(P<0.01, P<0.05), as well as increased DAI scores, colon histopathological scores, contents of serum pro-inflammatory factors TNF-α and IL-1ß, colonic iNOS/CD68 fluorescence positive expression, iNOS protein and mRNA expressions(P<0.01). Compared with the model group, the above indicators were significantly improved in rats of the EA group and the western medication group(P<0.01, P<0.05). CONCLUSIONS: EA of "Changbing Decoction" can improve UC of rats by regulating the polarization of colonic macrophages, inhibiting the generation of M1 macrophages and promoting the generation of M2 macrophages.

Lonicerae Japonicae Caulis: a review of its research progress of active metabolites and pharmacological effects.

Lonicerae Japonicae Caulis is the aboveground stem part of the Lonicera Japonica Thunb, which belongs to the medicine food homology species in China. It has the effects of clearing away heat, toxic material, dredging wind and unblocking collaterals. Modern research shows that it contains various active metabolites and a wide range of pharmacological effects, which is of great research and clinical application value. It mainly contains organic acids, volatile oils, flavonoids, triterpenes, triterpene saponins and other active metabolites. Its pharmacological effects mainly include anti-inflammatory, antibacterial, antitumor, antioxidant, and repairing bone and soft tissue. Based on the literature reports in recent years, the active metabolites, pharmacological effects and mechanisms of Lonicerae Japonicae Caulis were sorted out and summarized. It lays a foundation for explaining the efficacy material basis and application value of Lonicerae Japonicae Caulis. It aims to provide a reference for the in-depth research, development and utilization of Lonicerae Japonicae Caulis.

[Effect of electroacupuncture on colonic autophagy and AMPK/mTOR signaling pathway in rats with acute ulcerative colitis].

OBJECTIVE: To observe the effect of electroacupuncture （EA） at "Zhongwan" （CV12）, "Tianshu" （ST25） and "Shangjuxu" （ST37） （an acupoint prescription "Changbingfang" for treatment of intestinal disorders） on autophagy and expression of AMPK/mTOR signaling pathway in rats with ulcerative colitis （UC）, so as to explore its mechanism underlying improvement of UC. METHODS: Thirty-two male SD rats were randomly divided into control, model, medication and EA groups, with 8 rats in each group. The UC model was established by free drinking of 5% dextran sulfate sodium salt solution for 7 days. EA stimulation （10 Hz/50 Hz） was delivered to CV12, ST25 and ST37 for 20 min, once a day for 3 consecutive days. Rats of the medication group received gavage of mesalazine suspension （200 mg/kg） once a day, 3 times in total. The rats' general conditions were recorded for calculating the disease activity index （DAI） score （0-4 points）. Histomorphological changes of colon were observed via HE staining. The levels of serum interleukin 6 （IL-6）, tumor necrosis factor-α （TNF-α） and IL-10 were measured by ELISA. The mRNA expressions of LC3B and p62 were tested by fluorescence quantitative PCR. Western blot was used to detect the expression levels of LC3B, p62 and AMPK/mTOR pathway related proteins in colon tissues. RESULTS: Compared with the control group, the DAI score, contents of serum IL-6 and TNF-α, the expression levels of p62 protein and mRNA, ratio of p-mTOR/mTOR were significantly increased （P<0.01）； while the content of serum IL-10, the expression levels of LC3B mRNA, ratio of LC3BⅡ/LC3BⅠ and p-AMPK/AMPK were decreased （P<0.01, P<0.05） in the model group. Relevant to the model group, modeling-induced increases of DAI score, serum IL-6, TNF-α and IL-10 contents, expressions of p62 protein and mRNA, LC3B mRNA, ratio of p-mTOR/mTOR, LC3BⅡ/LC3BⅠ and p-AMPK/AMPK were reversed in both medication and EA groups （P<0.01, P<0.05）. The effect of EA was apparently superior to that of mesalazine in up-regulating ratio of LC3BⅡ/LC3BⅠ and p-AMPK/AMPK, p62 mRNA expression （P<0.01, P<0.05）, and in down-regulating ratio of p-mTOR/mTOR （P<0.05）. H.E. staining showed severe damage of the colonic mucosal barrier with infiltration of a large number of inflammatory cells in the model group, which was milder in medication and EA groups. CONCLUSION: EA of acupoint recipe "Changbingfang" can improve the symptoms in UC rats, which may be related to its functions in promoting colonic autophagy, increasing AMPK phosphorylation level, and decreasing mTOR phosphorylation level.

Deleted in lymphocytic leukemia 2 (DLEU2): a possible biomarker that holds promise for future diagnosis and treatment of cancer.

The mechanism of deleted in lymphocytic leukemia 2 (DLEU2)-long non-coding RNA in tumors has become a major point of interest in recent research related to the occurrence and development of a variety of tumors. Recent studies have shown that the long non-coding RNA DLEU2 (lncRNA-DLEU2) can cause abnormal gene or protein expression by acting on downstream targets in cancers. At present, most lncRNA-DLEU2 play the role of oncogenes in different tumors, which are mostly associated with tumor characteristics, such as proliferation, migration, invasion, and apoptosis. The data thus far show that because lncRNA-DLEU2 plays an important role in most tumors, targeting abnormal lncRNA-DLEU2 may be an effective treatment strategy for early diagnosis and improving the prognosis of patients. In this review, we integrated lncRNA-DLEU2 expression in tumors, its biological functions, molecular mechanisms, and the utility of DLEU2 as an effective diagnostic and prognostic marker of tumors. This study aimed to provide a potential direction for the diagnosis, prognosis, and treatment of tumors using lncRNA-DLEU2 as a biomarker and therapeutic target.

A HER2-targeted antibody-novel DNA topoisomerase I inhibitor conjugate induces durable adaptive antitumor immunity by activating dendritic cells.

We designed and developed a novel DNA topoisomerase I inhibitor MF-6, which was a more potent cytotoxin and a more potent inducer of immunogenic cell death compared with DXd. To utilize MF-6's ability to induce antitumor immunity, a human epidermal growth factor receptor 2 (HER2)-targeted antibody-drug conjugate (ADC) trastuzumab-L6 that included a cleavable linker and MF-6 was developed. Different from traditional cytotoxic ADC, the antitumor activity of trastuzumab-L6 was assessed by inducing tumor cell immunogenic cell death, activating dendritic cells and cytotoxic CD8+ T cells to acquire durable adaptive immune memory. Tumor cells treated with trastuzumab-L6 were committed to immunogenic cell death, with upregulation of damage-associated molecular patterns and antigen presentation molecules. In a syngeneic tumor model with a mouse cell line that expressed human HER2, immunocompetent mice showed greater antitumor efficacy compared with nude mice. The trastuzumab-L6-cured immunocompetent mice acquired adaptive antitumor memory and rejected subsequent tumor cell challenge. The trastuzumab-L6 efficacy was abrogated when cytotoxic CD8+ T cells were depleted and enhanced when regulatory CD4+ T cells were depleted. The combination of trastuzumab-L6 with immune checkpoint inhibitors significantly increased antitumor efficacy. Enhanced T cell infiltration, dendritic cell activation, and decreased type M2 macrophages in tumor post trastuzumab-L6 administration confirmed the immune-activating responses. In conclusion, trastuzumab-L6 was considered to be an immunostimulatory agent, rather than a traditional cytotoxic ADC, and its antitumor efficacy was enhanced when combined with an anti-PD-L1 and anti-CTLA-4 antibody, which suggested a potential therapeutic strategy.

Preclinical Evaluation of 9MW2821, a Site-Specific Monomethyl Auristatin E-based Antibody-Drug Conjugate for Treatment of Nectin-4-expressing Cancers.

Overexpression of nectin cell adhesion protein 4 correlates with cancer progression and poor prognosis in many human malignancies. Enfortumab vedotin (EV) is the first nectin-4-targeting antibody-drug conjugate (ADC) approved by the FDA for the treatment of urothelial cancer. However, inadequate efficacy has limited progress in the treatment of other solid tumors with EV. Furthermore, ocular, pulmonary, and hematologic toxic side effects are common in nectin-4-targeted therapy, which frequently results in dose reduction and/or treatment termination. Thus, we designed a second generation nectin-4-specific drug, 9MW2821, based on interchain-disulfide drug conjugate technology. This novel drug contained a site specifically conjugated humanized antibody and the cytotoxic moiety monomethyl auristatin E. The homogenous drug-antibody ratio and novel linker chemistry of 9MW2821 increased the stability of conjugate in the systemic circulation, enabling highly efficient drug delivery and avoiding off-target toxicity. In preclinical evaluation, 9MW2821 exhibited nectin-4-specific cell binding, efficient internalization, bystander killing, and equivalent or superior antitumor activity compared with EV in both cell line-derived xenograft and patient-derived xenograft (PDX) models. In addition, 9MW2821 demonstrated a favorable safety profile; the highest nonseverely toxic dose in monkey toxicologic studies was 6 mg/kg, with milder adverse events compared with EV. Overall, 9MW2821 is a nectin-4-directed, investigational ADC based on innovative technology that endowed the drug with compelling preclinical antitumor activity and a favorable therapeutic index. The 9MW2821 ADC is being investigated in a phase I/II clinical trial (NCT05216965 and NCT05773937) in patients with advanced solid tumors.

Promising candidates from drug clinical trials: Implications for clinical treatment of Alzheimer's disease in China.

Alzheimer's disease is the most common neurodegenerative disease. Prior to 2017, National Medical Products Administration approved only four drugs to treat Alzheimer's disease, including three cholinesterase inhibitors and one N-methyl-D-aspartate receptor antagonist. We queried ClinicalTrials.gov to better understand Alzheimer's drug development over the past 5 years and found 16 promising candidates that have entered late-stage trials and analyzed their impact on clinical treatment of Alzheimer's disease in China. The 16 compounds selected include disease-modifying therapies and symptomatic therapies. The research and development pipeline now focuses on disease-modifying therapies such as gantenerumab, aducanumab, ALZ-801, ALZT-OP1, donanemab, lecanemab, simufilam, NE3107, semaglutide, and GV-971, which could put an end to the situation where Alzheimer's patients in China have no effective treatment alternatives. The reuse of drugs or combinations currently under investigation for the psychiatric treatment of Alzheimer's disease, including AXS-05, AVP-786, nabilone, brexpiprazole, methylphenidate, and pimavanserin, could provide physicians with additional treatment options. Although most of these drugs have not been explored in China yet, due to the current development trend in this field in China, it is expected that China will be involved in research on these drugs in the future.

Epigenetic mechanisms modulate thyroid transcription factor 1-mediated transcription of the surfactant protein B gene.

Epigenetic regulation of transcription plays an important role in cell-specific gene expression by altering chromatin structure and access of transcriptional regulators to DNA binding sites. Surfactant protein B (Sftpb) is a developmentally regulated lung epithelial gene critical for lung function. Thyroid transcription factor 1 (Nkx2-1) regulates Sftpb gene expression in various species. We show that Nkx2-1 binds to the mouse Sftpb (mSftpb) promoter in the lung. In a mouse lung epithelial cell line (MLE-15), Nkx2-1 knockdown reduces Sftpb expression, and mutation of Nkx2-1 cis-elements significantly reduces mSftpb promoter activity. Whether chromatin structure modulates Nkx2-1 regulation of Sftpb transcription is unknown. We found that DNA methylation of the mSftpb promoter inversely correlates with known patterns of Sftpb expression in vivo. The mSftpb promoter activity can be manipulated by altering its cytosine methylation status in vitro. Nkx2-1 activation of the mSftpb promoter is impaired by DNA methylation. The unmethylated Sftpb promoter shows an active chromatin structure enriched in the histone modification H3K4me3 (histone 3-lysine 4 trimethylated). The ATP-dependent chromatin remodeling protein Brg1 is recruited to the Sftpb promoter in Sftpb-expressing, but not in non-expressing tissues and cell lines. Brg1 knockdown in MLE-15 cells greatly decreases H3K4me3 levels at the Sftpb promoter region and expression of the Sftpb gene. Brg1 can be co-immunoprecipitated with Nkx2-1 protein. Last, Nkx2-1 and Brg1 with intact ATPase activity are required for mSftpb promoter activation in vitro. Our findings suggest that DNA methylation and chromatin modifications cooperate with Nkx2-1 to regulate Sftpb gene cell specific expression.

Precise detection of a murine germline mutation of the Notch3 gene associated with kyphosis and developmental disorders.

OBJECTIVE: Humpback (hpbk) mice harbor a pathogenic mutation in the Notch3 gene and can serve as a beneficial animal model for investigating human myopathy, kyphosis, and developmental disorders, including lateral meningocele syndrome. Detection of the point mutation in hpbk mice is important for maintaining strains and scrutinizing genetic rescues, especially considering that homozygous mice are infertile and indistinguishable from their littermates at a young age. This study aimed for the development of a novel, precise, and time-saving genotyping method to identify the mutation in hpbk mice. MATERIALS AND METHODS: In order to study the hpbk mouse line, we describe how we applied several tools, including quantitative polymerase chain reaction (qPCR), multiplex tetra-primer amplification-refractory mutation system (ARMS-PCR) and Sanger sequencing, toward the recognition of heterozygous and homozygous mice. RESULTS: The Notch3 mutation was clearly identified using qPCR and ARMS assays, but the latter was a more precise and cost-effective approach. The lengths of the ARMS-PCR amplicons are 210 bp and 164 bp for the wild-type and hpbk alleles, respectively. Moreover, the genotyping results for each mouse were corroborated by Sanger DNA sequencing. CONCLUSION: Our newly developed PCR-based ARMS system affords a swift and precise way to genotype the hpbk mice. ARMS-PCR does not rely on any advanced equipment and is useful as a genotyping method for other model organisms that harbor a pathogenic variant.