ObjectiveBased on transcriptomics， to explore the mechanism of Hei Xiaoyaosan regulating the phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） signaling pathway to improve the learning and memory ability of insomnia rats with liver depression syndrome. MethodsSixty 8-week-old male SD rats were randomly divided into the blank group， model group， eszopiclone group （0.09 mg·kg^-1）， and low， medium， and high dose groups of Hei Xiaoyaosan （3.82， 7.65， 15.30 g·kg^-1）， with ten rats in each group. Except for the blank group， the other groups were induced insomnia rat model with liver depression by chronic restraint， tail clamping stimulation and intraperitoneal injection of p-chlorophenylalanine （PCPA）. Each treatment group received intragastric administration according to the specified dosage， once a day for 14 consecutive days. The pentobarbital sodium cooperative sleep test， open field test， and Morris water maze test were used to test the sleep quality， depressive-like behavior， and learning and memory abilities of rats. Additionally， enzyme-linked immunosorbent assay （ELISA） was used to detect the contents of 5-hydroxytryptamine （5-HT）， γ-aminobutyric acid （GABA）， brain-derived neurotrophic factor （BDNF）， glial cell line-derived neurotrophic factor （GDNF） and nitric oxide （NO） in hippocampus. Hematoxylin-eosin （HE） staining was performed to observe pathological changes of the hippocampal tissue， while terminal deoxynucleotidyl transferase deoxyuridine triphosphate （dUTP） nick end labeling （TUNEL） was used to evaluate apoptosis of hippocampal neurons. Transcriptomic sequencing technology was employed to identify differentially expressed genes in hippocampus between the model group and the blank group， as well as between the medium-dose group of Hei Xiaoyaosan and the model group. Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis were performed on the intersecting genes. Subsequently， the enriched key genes and signaling pathways were analyzed and verified. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was utilized to assess the mRNA expression levels of phosphatase and tensin homolog （PTEN）， B-cell lymphoma-2 （Bcl-2）-like protein 11 （BCL2L11）， and mitogen-activated protein kinase 1 （MAPK1） in hippocampus， and Western blot was employed to evaluate the protein expressions of PI3K， phosphorylation （p）-PI3K， Akt， p-Akt， Bcl-2， Bcl-2-associated X protein （Bax）， and cleaved Caspase-3 in the same tissue. ResultsCompared with the blank group， the model group exhibited a reduction in body weight， an increase in sleep latency， and a decrease in sleep duration （P<0.01）. Additionally， rats showed obvious depression-like behavior， and their learning and memory abilities decreased. Furthermore， the contents of 5-HT， GABA， NO， BDNF and GDNF in hippocampus decreased （P<0.01）. Histological examination revealed a disorganized cell arrangement in the CA1 region of the hippocampus， characterized by irregular cell shapes， a reduced cell count， deeply stained and pyknotic nuclei， increased vacuolar degeneration， and an elevated apoptosis rate （P<0.01）. Compared with the model group， the body weight of the high and medium dose groups of Hei Xiaoyaosan increased， the sleep latency shortened and the sleep time prolonged （P<0.05， P<0.01）. Additionally， depression-like behavior and learning and memory abilities of rats were significantly improved， the levels of 5-HT， GABA， NO， BDNF and GDNF in the hippocampus increased （P<0.05， P<0.01）. These interventions also ameliorated pathological damage in the hippocampal CA1 area and reduced the apoptosis of hippocampal neurons （P<0.01）. Transcriptomic sequencing results indicated that Hei Xiaoyaosan might exert a therapeutic effect by regulating PI3K/Akt pathway through key mRNAs such as PTEN， BCL2L11， and MAPK1. The roles of these key mRNAs and proteins within PI3K/Akt pathway were further validated. In comparison to the blank group， the expression levels of PTEN， BCL2L11 and MAPK1 mRNA in the hippocampus of rats in the model group were increased （P<0.01）， while the protein expression levels of p-PI3K， p-Akt and Bcl-2 were decreased （P<0.01）， and the protein expression levels of PTEN， Bax and cleaved Caspase-3 were increased （P<0.01）. Compared with the model group， the high-dose and medium-dose groups of Hei Xiaoyaosan could down-regulate the expressions of PTEN， BCL2L11 and MAPK1 mRNAs （P<0.01）， up-regulate the expressions of p-PI3K， p-Akt and Bcl-2 proteins （P<0.01）， and down-regulate the protein expressions of PTEN， Bax and cleaved Caspase-3 （P<0.05， P<0.01）. ConclusionHei Xiaoyaosan may regulate PI3K/Akt signaling pathway by down-regulating expressions of key genes such as PTEN， BCL2L11 and MAPK1， and thus improve the learning and memory abilities of insomnia rats with liver depression syndrome.

The Janus kinase/signal transducers and activators of transcription (JAK-STAT) control natural killer (NK) cells development and cytotoxic functions, however, whether long non-coding RNAs (lncRNAs) are involved in this pathway remains unknown. We found that miR155HG was elevated in activated NK cells and promoted their proliferation and effector functions in both NK92 and induced-pluripotent stem cells (iPSCs)-derived NK (iPSC-NK) cells, without reliance on its derived miR-155 and micropeptide P155. Mechanistically, miR155HG bound to miR-6756 and relieved its repression of JAK3 expression, thereby promoting the JAK-STAT pathway and enhancing NK cell proliferation and function. Further investigations disclosed that upon cytokine stimulation, STAT3 directly interacts with miR155HG promoter and induces miR155HG transcription. Collectively, we identify a miR155HG-mediated positive feedback loop of the JAK-STAT signaling. Our study will also provide a power target regarding miR155HG for improving NK cell generation and effector function in the field of NK cell adoptive transfer therapy against cancer, especially iPSC-derived NK cells.

Obesity usually exacerbates the immunosuppressive tumor microenvironment (ITME), hindering CD8⁺ T cell infiltration and function, which further represents a significant barrier to the efficacy of immunotherapy. Herein, a multifunctional liposomal system (CR-Lip) for encapsulating celastrol (CEL) was utilized to remodel obesity-related ITME and improve cancer immunotherapy, wherein Ginsenoside Rg3 (Rg3) was detected interspersed in the phospholipid bilayer and its glycosyl exposed on the surface of the liposome. CR-Lip had a relatively uniform size (116.5 nm), facilitating favorable tumor tissue accumulation through the interaction between Rg3 and glucose transporter 1 overexpressed in obese tumor cells. Upon reaching the tumor region, CR-Lip was found to induce the immunogenic cell death (ICD) of HFD tumor cells. Notably, the level of PHD3 in HFD tumor cells was effectively boosted by CR-Lip to effectively block metabolic reprogramming and increase the availability of major free fatty acids fuel sources. In vivo, experiments studies revealed that the easy-obtained nano platform stimulated enhanced the production of various cytokines in tumor tissues, DC maturation, CD8⁺ T-cell infiltration, and synergistic anticancer therapeutic potency with aPD-1 (tumor inhibition rate = 82.1%) towards obesity-related melanoma. Consequently, this study presented an efficacious approach to tumor immunotherapy in obese mice by encompassing tumor eradication, inducing ICD, and reprogramming metabolism. Furthermore, it offered a unique insight into a valuable attempt at the immunotherapy of obesity-associated related tumors.

Growth arrest DNA damage-inducible protein 45 β (GADD45B) has been reported to be a regulatory factor for active DNA demethylation and is implicated in the modulation of synaptic plasticity and chronic stress-related psychopathological processes. However, its precise role and mechanism of action in stress susceptibility remain elusive. In this study, we found a significant reduction in GADD45B expression specifically in the ventral, but not the dorsal hippocampal CA1 (dCA1) of stress-susceptible mice. Furthermore, we demonstrated that GADD45B negatively regulates susceptibility to social stress and NMDA receptor-dependent long-term potentiation (LTP) in the ventral hippocampal CA1 (vCA1). Importantly, through pharmacological inhibition using the NMDA receptor antagonist MK801, we provided further evidence supporting the hypothesis that GADD45B potentially modulates susceptibility to social stress by influencing NMDA receptor-mediated LTP. Collectively, these results suggested that modulation of NMDA receptor-mediated synaptic plasticity is a pivotal mechanism underlying the regulation of susceptibility to social stress by GADD45B.
Animals ; Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors* ; CA1 Region, Hippocampal/drug effects* ; Male ; Stress, Psychological/physiopathology* ; Mice ; Neuronal Plasticity/drug effects* ; Long-Term Potentiation/drug effects* ; Mice, Inbred C57BL ; Antigens, Differentiation/metabolism* ; Dizocilpine Maleate/pharmacology* ; Excitatory Amino Acid Antagonists/pharmacology* ; GADD45 Proteins

ObjectiveBased on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， network pharmacology and pharmacodynamics， to investigate the pharmacodynamic material basis and mechanism of Hei Xiaoyaosan in improving learning and memory ability of insomnia rats. MethodUPLC-Q-TOF-MS was used to characterize the chemical constituents of Hei Xiaoyaosan. Network pharmacology was applied to construct the network of active ingredients-intersecting targets-pathways， and molecular docking was performed on key ingredients and core targets. Sixty 8-week-old male SD rats were selected and randomly divided into blank group， model group， Hei Xiaoyaosan low， medium， and high dose groups（3.82， 7.65， 15.30 g·kg^-1）， and zolpidem tartrate group（0.5 mg·kg^-1）， with 10 rats in each group. Except for the blank group， the insomnia model was induced by intraperitoneal injection of p-chlorophenylalanine（PCPA） for 4 consecutive days. Rats in each dosing group were administered the corresponding dose by gavage， once a day for 14 consecutive days. Morris water maze test was utilized to assess the learning and memory ability of rats， transmission electron microscopy was employed to examine the ultrastructure of hippocampal synapses， enzyme-linked immunosorbent assay（ELISA） was conducted to analyze the levels of 5-hydroxytryptamine（5-HT）， interleukin-6（IL-6）， and tumor necrosis factor-α（TNF-α） in hippocampal tissues， and Western blot was performed to detect the expression levels of tumor suppressor protein p53（TP53）， rat sarcoma virus（RAS）， epidermal growth factor receptor（EGFR）， cyclic adenosine monophosphate（cAMP）-response element binding protein（CREB） binding protein（CREBBP）， glycogen synthase kinase-3β（GSK-3β）， protein kinase B1（Akt1）， nitric oxide synthase 1（NOS1）， phosphorylated（p）-Akt1， and p-GSK-3β in hippocampal tissues. Additionally， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to assess the mRNA expression levels of TP53， RAS， EGFR， CREBBP， GSK-3β， Akt1 and NOS1. ResultA total of 176 components were identified in Hei Xiaoyaosan， mainly flavonoids， triterpene saponins， phenylpropanoids and other compounds. Network pharmacological analysis revealed that TP53， V-Ha-Ras Harvey Rat sarcoma viral oncogene homolog（HRAS）， neuroblastoma sarcoma viral oncogene homolog（NRAS）， EGFR， CREBBP， GSK-3β， Akt1 and NOS1 were the key targets of Hei Xiaoyaosan in treating insomnia. The core targets were predominantly associated with cAMP， RAS， Ras-associated protein 1（Rap1）， advanced glycation end products（AGE）/receptor for AGE（RAGE）， and EGFR signaling pathways， and the key active ingredients of Hei Xiaoyaosan in treating insomnia were 8-shogaol， ligustilide F， 6-gingerol， levistilide A and senkyunolide E. Animal experiment results demonstrated that Hei Xiaoyaosan medium and high dose groups significantly increased body weight， shortened sleep latency and prolonged sleep duration in insomnia rats（P<0.01）， significantly decreased escape latency and increased platform crossing frequency（P<0.01）， and improved the pathological changes of hippocampal synaptic ultrastructure. Meanwhile， the two groups could significantly elevate 5-HT level， Akt1 mRNA expression， Akt1 and p-Akt1 protein expression（P<0.01）， reduce inflammatory factor levels（P<0.01）， and down-regulate protein expression levels of TP53， RAS， NOS1， EGFR， CREBBP， GSK-3β and p-GSK-3β（P<0.01）， as well as mRNA expression levels of TP53， RAS， NOS1， EGFR， CREBBP and GSK-3β in hippocampal tissues（P<0.01）. ConclusionThis study determined that the five key active ingredients（8-shogaol， ligustilide F， 6-gingerol， levistilide A and senkyunolide E） in Hei Xiaoyaosan may improve the learning and memory ability of insomnia rats by regulating signaling pathways such as cAMP， RAS， and EGFR， providing an important reference for its mechanism research and clinical application.

ObjectiveTo develop traditional Chinese medicine （TCM） formulae for the treatment of nonsevere coronavirus disease 2019 （COVID-19） and to explore its anti-inflammatory mechanism. MethodsThe dysregulated signaling pathways were determined in macrophages from bronchoalveolar lavage fluid of COVID-19 patients and in lung epithelial cells infected with SARS-CoV-2 in vitro based on transcriptome analysis. A total of 102 TCM formulae for the clinical treatment of nonsevere COVID-19 were collected through literature. The pathway-reversing rates of these formulae in macrophages and lung epithelial cells were evaluated based on signature signaling pathways， and the basic formula was determined in conjunction with TCM theory. The commonly used Chinese materia medica for nonsevere COVID-19 were summarized from the 102 TCM formulae as abovementioned. And together with the screening results from the Pharmacopoeia of the People's Republic of China， a “Chinese materia medica pool” was esta-blished for the development of TCM formulae for COVID-19. The regulatory effects of each herb on signaling pathways were obtained based on targeted transcriptome analysis. Oriented at reversing dysregulated signaling pathways of COVID-19， the calculation was carried out， and the artificial intelligent methods for compositing formulae， that are exhaustive method and parallel computing， were used to obtain candidate compound formulas. Finally， with reference to professional experience， an innovative formula for the treatment of nonsevere COVID-19 was developed. The ethanol extract of the formula was evaluated for its anti-inflammatory effects by detecting the mRNA expression of interleukin 1b （Il1b）， C-X-C motif chemokine ligand 2 （Cxcl2）， C-X-C motif chemokine ligand 10 （Cxcl10）， C-C motif chemokine ligand 2 （Ccl2）， nitric oxide synthase 2 （Nos2）， and prostaglandin-endoperoxide synthase 2 （Ptgs2） using reverse transcription-quantitative polymerase chain reaction （RT-qPCR） in RAW264.7 cells treated with lipopolysaccharide （LPS）. ResultsIn macrophages and lung epithelial cells， 34 dysregulated signaling pathways associated with COVID-19 were identified respectively. The effects of the 102 formulae for clinical treatment of nonsevere COVID-19 were evaluated based on the dysregulated signaling pathways and targeted transcriptome， and the result showed that Yinqiao Powder and Pingwei Powder （银翘散合平胃散， YQPWP） ranked first， reversing 91.18% of the dysregulated signaling pathways in macrophages and 100% of the dysregulated signaling pathways in lung epithelial cells. Additionally， YQPWP had the function of scattering wind and clearing heat， resolving toxins and removing dampness in accordance with the pathogenesis of wind-heat with dampness in COVID-19. It was selected as the basic formula， and was further modified and optimized to develop an innovative fomula Qiaobang Zhupi Yin （翘蒡术皮饮， QBZPY） based on expert experience and artificial intelligence in composing formulae. QBZPY can reverse all the dysregulated signaling pathways associated with COVID-19 in macrophages and lung epithelial cells， with the reversing rates of 100%. The chief medicinal of QBZPY， including Lianqiao （Fructus Forsythiae）， Xixiancao （Herba Siegesbeckiae） and Niubangzi （Fructus Arctii）， can down-regulate multiple signaling pathways related with virus infection， immune response， and epithelial damage. RT-qPCR results indicated that compared with the model group， the QBZPY group down-regulated the mRNA expression of Il1b， tumor necrosis factor （Tnf）， Cxcl2， Cxcl10， Ccl2， Nos2 and Ptgs2 induced by LPS in RAW264.7 cells （P＜0.05 or P＜0.01）. ConclusionBased on targeted transcriptome analysis， expert experience in TCM and artificial intelligence， QBZPY has been developed for the treatment of nonsevere COVID-19. The ethanol extract of QBZPY has been found to inhibit mRNA expression of several pro-inflammatory genes in a cellular inflammation model.

Objective To study the effect of neuromodulatory protein-1(NRG-1)in inhibiting sepsis induced myocardial injury and its mechanism.Methods The rat sepsis model was established by cecal ligation and puncture(CLP).SD rats were divided into the sham operation group,sepsis group,sepsis+NRG group(rhNRG,10 μg/kg).After 12,24 h of successful modeling,the heart and peripheral serum of the surviving rats in each group were taken respectively.The HE staining was used to observe the changes of cardiac tissue morphology and structure,and ELISA was used to detect the expression levels of creatine kinase(CK),crea-tine kinase MB isoenzyme(CK-MB),sensitive troponin Ⅰ(cTnⅠ)in serum,tumor necrosis factor-α(TNF-α)in cardiac tissue and IL-6 expression level;Western blot was used to detect the phosphorylation protein ki-nase B(p-Akt),phosphorylation glycogen synthase kinaseβ(p-GSK3β),B-cell lymphoma/leukemia-2(Bcl-2)and Bax protein expression in rat myocardial tissue.Results After 12,24 h of modeling,compared with the sham group,the expression levels of CK,CK-MB and cTnⅠ in serum,TNF-α,IL-6 and Bax protein in myocar-dial tissue in the sepsis group all were significantly increased(P＜0.05),while the expression levels of p-Akt and p-GSK3β in myocardial tissue were significantly decreased(P＜0.05).After 12,24 h of modeling,com-pared with the sepsis group,the expression levels of CK,CK-MB,cTn Ⅰ in the serum and the expression levels of TNF-α,IL-6 in the myocardial tissue of the sepsis+NRG group were significantly decreased;after 24 h of modeling,compared with the sepsis group,the expression level of Bax protein in myocardial tissue of the sep-sis+NRG group was decreased,while the p-Akt,p-GSK3β expression levels were increased(P＜0.05).The pathological results showed that compared with the sham operation group,the sepsis group produced signifi-cant lesions;compared with the sepsis group,the lesions in the sepsis+NRG group were alleviated.Conclusion The expression levels of related biomarkers in septic myocardial injury have change.NRG-1 could improve the cardiac function through Akt/GSK3β pathway,inhibit the related proinflammatory factors and reduce the myocardial tissue damage.

Objective:To further improve the understanding of paroxysmal nocturnal hemoglobinuria (PNH), we retrospectively analyzed and summarized the clinical characteristics, treatment status, and survival status of patients with PNH in Zhejiang Province.Methods:This study included 289 patients with PNH who visited 20 hospitals in Zhejiang Province. Their clinical characteristics, comorbidity, laboratory test results, and medications were analyzed and summarized.Results:Among the 289 patients with PNH, 148 males and 141 females, with a median onset age of 45 (16-87) years and a peak onset age of 20-49 years (57.8% ). The median lactic dehydrogenase (LDH) level was 1 142 (604-1 925) U/L. Classified by type, 70.9% (166/234) were classical, 24.4% (57/234) were PNH/bone marrow failure (BMF), and 4.7% (11/234) were subclinical. The main clinical manifestations included fatigue or weakness (80.8%, 235/289), dizziness (73.4%, 212/289), darkened urine color (66.2%, 179/272), and jaundice (46.2%, 126/270). Common comorbidities were hemoglobinuria (58.7% ), renal dysfunction (17.6% ), and thrombosis (15.0% ). Moreover, 82.3% of the patients received glucocorticoid therapy, 70.9% required blood transfusion, 30.7% used immunosuppressive agents, 13.8% received anticoagulant therapy, and 6.3% received allogeneic hematopoietic stem cell transplantation. The 10-year overall survival (OS) rate was 84.4% (95% CI 78.0% -91.3% ) . Conclusion:Patients with PNH are more common in young and middle-aged people, with a similar incidence rate between men and women. Common clinical manifestations include fatigue, hemoglobinuria, jaundice, renal dysfunction, and recurrent thrombosis. The 10-year OS of this group is similar to reports from other centers in China.

Objective:To evaluate the efficacy and safety of hypomethylating agents (HMA) in patients with myelodysplastic syndromes (MDS) .Methods:A total of 409 MDS patients from 45 hospitals in Zhejiang province who received at least four consecutive cycles of HMA monotherapy as initial therapy were enrolled to evaluate the efficacy and safety of HMA. Mann-Whitney U or Chi-square tests were used to compare the differences in the clinical data. Logistic regression and Cox regression were used to analyze the factors affecting efficacy and survival. Kaplan-Meier was used for survival analysis. Results:Patients received HMA treatment for a median of 6 cycles (range, 4-25 cycles) . The complete remission (CR) rate was 33.98% and the overall response rate (ORR) was 77.02%. Multivariate analysis revealed that complex karyotype ( P=0.02, OR=0.39, 95% CI 0.18-0.84) was an independent favorable factor for CR rate. TP53 mutation ( P=0.02, OR=0.22, 95% CI 0.06-0.77) was a predictive factor for a higher ORR. The median OS for the HMA-treated patients was 25.67 (95% CI 21.14-30.19) months. HMA response ( P=0.036, HR=0.47, 95% CI 0.23-0.95) was an independent favorable prognostic factor, whereas complex karyotype ( P=0.024, HR=2.14, 95% CI 1.10-4.15) , leukemia transformation ( P<0.001, HR=2.839, 95% CI 1.64-4.92) , and TP53 mutation ( P=0.012, HR=2.19, 95% CI 1.19-4.07) were independent adverse prognostic factors. There was no significant difference in efficacy and survival between the reduced and standard doses of HMA. The CR rate and ORR of MDS patients treated with decitabine and azacitidine were not significantly different. The median OS of patients treated with decitabine was longer compared with that of patients treated with azacitidine (29.53 months vs 20.17 months, P=0.007) . The incidence of bone marrow suppression and pneumonia in the decitabine group was higher compared with that in the azacitidine group. Conclusion:Continuous and regular use of appropriate doses of hypomethylating agents may benefit MDS patients to the greatest extent if it is tolerated.

Breast cancer, a malignant tumor with a high incidence in women, lacks in vitro research models that can represent the biological functions of breast tumors in vivo. As a new biological tool, the organoid model has unique advantages over traditional methods, such as cell culture and patient-derived xenografts. Combining organoids with other emerging technologies, such as gene engineering and microfluidic chip technology, provides an effective method to compensate for the deficiencies in organoid models of breast cancer in vivo. The emergence of breast cancer organoids has provided new tools and research directions in precision medicine, and drug research. In this review, we summarized the merits and demerits of organoids compared to traditional biological models, explored the latest developments in the combination of new technologies and organoid models, and discussed the construction methods and application prospects of different breast cancer organoid models.