Decoction is the most commonly used dosage form in the clinical treatment of traditional Chinese medicine (TCM). During boiling, the violent movement of various active ingredients in TCM creates molecular forces such as hydrogen bonding, π-π stacking, hydrophobic interactions and electrostatic interactions, which results in the formation of self-assembled aggregates in decoction (SADs), including particles, gels, fibers, etc. It was found that SADs widely existed in decoction with biological activities superior to both effective monomers and their physical mixtures, providing a new idea to reveal the pharmacodynamic material basis of Chinese herbal medicine from the perspective of component interactions-phase structure. Recently, SADs have become a novel focus of research in TCM. This paper reviewed their relevant studies in recent years and found some issues to be concerned in the research, such as the polydispersity of decoction system, instability of active ingredient interactions during boiling, uncertainty of the aggregates self-assembly rules, and stability, purity, yield of the products. In this regard, some solutions and new ideas were presented for the integrated development and clinical application of SADs.

Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw, and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamHⅠand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 β-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel β-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.
Humans ; Enterotoxins/genetics* ; Superantigens/genetics* ; Catalytic Domain ; Selenoprotein W/metabolism* ; Receptors, Antigen, T-Cell

Background and Objectives: Osteoblasts are derived from bone marrow mesenchymal stem cells (BMMSCs) and playimportant role in bone remodeling. While our previous studies have investigated the cell subtypes and heterogeneity in osteoblasts and BMMSCs separately, cell-to-cell communications between osteoblasts and BMMSCs in vivo in humans have not been characterized. The aim of this study was to investigate the cellular communication between human primary osteoblasts and bone marrow mesenchymal stem cells. Methods: and Results: To investigate the cell-to-cell communications between osteoblasts and BMMSCs and identifynew cell subtypes, we performed a systematic integration analysis with our single-cell RNA sequencing (scRNA-seq) transcriptomes data from BMMSCs and osteoblasts. We successfully identified a novel preosteoblasts subtype which highly expressed ATF3, CCL2, CXCL2 and IRF1. Biological functional annotations of the transcriptomes suggested that the novel preosteoblasts subtype may inhibit osteoblasts differentiation, maintain cells to a less differentiated status and recruit osteoclasts. Ligand-receptor interaction analysis showed strong interaction between mature osteoblasts and BMMSCs. Meanwhile, we found FZD1 was highly expressed in BMMSCs of osteogenic differentiation direction. WIF1 and SFRP4, which were highly expressed in mature osteoblasts were reported to inhibit osteogenic differentiation. We speculated that WIF1 and sFRP4 expressed in mature osteoblasts inhibited the binding of FZD1 to Wnt ligand in BMMSCs, thereby further inhibiting osteogenic differentiation of BMMSCs. Conclusions Our study provided a more systematic and comprehensive understanding of the heterogeneity of osteogenic cells. At the single cell level, this study provided insights into the cell-to-cell communications between BMMSCs and osteoblasts and mature osteoblasts may mediate negative feedback regulation of osteogenesis process.

This study aims to systematically evaluate the clinical efficacy and safety of Toutongning Capsules in the treatment of tension-type headache(TTH), so as to provide a corresponding basis for clinical treatment. Eight commonly used medical research databases and two clinical trial registration systems were retrieved with the time interval from the establishment of the database or system to November 2020. The randomized controlled trials of Toutongning Capsules in the treatment of TTH were screened out according to the pre-set criteria. The quality of the included papers was evaluated by the bias risk assessment tool in Cochrane Reviewers Handbook 6.1 and the data were statistically analyzed by RevMan v5.4 provided by Cochrane collaboration. A total of 13 studies were included and the quality of methodology was generally low. Meta-analysis showed that Toutongning Capsules assisted with western medicine therapy can effectively reduce the pain intensity(MD_(VAS)=-1.94,95%CI[-2.50,-1.38],P<0.000 01;MD_(NRS)=-0.83,95%CI[-0.86,-0.80],P<0.000 01), headache duration(SMD=-0.98,95%CI[-1.17,-0.79],P<0.000 01), headache frequency(MD=-1.01,95%CI[-1.16,-0.85],P<0.000 01), headache index(MD=-11.13,95%CI[-12.10,-10.16],P<0.000 01), anxiety and depression scale score(MD_(HAMA)=-4.02,95%CI[-6.58,-1.46],P=0.002;MD_(HAMD)=-2.67,95%CI[-4.04,-1.29],P=0.000 1), while Toutongning Capsules as monotherapy only reduced the headache score(MD=-2.24,95%CI[-2.97,-1.51],P<0.000 01). The available clinical studies demonstrate that Toutongning Capsules combined with western medicine in the treatment of TTH can improve the related outcome indicators, but the clinical safety and efficacy of Toutongning Capsules alone remain unclear. Due to the small number and low quality of the included studies, large-sample, multi-center, high-quality and strictly designed randomized controlled trials are still needed to verify the clinical efficacy in the future.
Capsules ; Databases, Factual ; Drugs, Chinese Herbal ; Humans ; Tension-Type Headache/drug therapy* ; Treatment Outcome

Objective: Several COVID-19 patients have overlapping comorbidities. The independent role of each component contributing to the risk of COVID-19 is unknown, and how some non-cardiometabolic comorbidities affect the risk of COVID-19 remains unclear. Methods: A retrospective follow-up design was adopted. A total of 1,160 laboratory-confirmed patients were enrolled from nine provinces in China. Data on comorbidities were obtained from the patients' medical records. Multivariable logistic regression models were used to estimate the odds ratio ( Results: Overall, 158 (13.6%) patients were diagnosed with severe illness and 32 (2.7%) had unfavorable outcomes. Hypertension (2.87, 1.30-6.32), type 2 diabetes (T2DM) (3.57, 2.32-5.49), cardiovascular disease (CVD) (3.78, 1.81-7.89), fatty liver disease (7.53, 1.96-28.96), hyperlipidemia (2.15, 1.26-3.67), other lung diseases (6.00, 3.01-11.96), and electrolyte imbalance (10.40, 3.00-26.10) were independently linked to increased odds of being severely ill. T2DM (6.07, 2.89-12.75), CVD (8.47, 6.03-11.89), and electrolyte imbalance (19.44, 11.47-32.96) were also strong predictors of unfavorable outcomes. Women with comorbidities were more likely to have severe disease on admission (5.46, 3.25-9.19), while men with comorbidities were more likely to have unfavorable treatment outcomes (6.58, 1.46-29.64) within two weeks. Conclusion Besides hypertension, diabetes, and CVD, fatty liver disease, hyperlipidemia, other lung diseases, and electrolyte imbalance were independent risk factors for COVID-19 severity and poor treatment outcome. Women with comorbidities were more likely to have severe disease, while men with comorbidities were more likely to have unfavorable treatment outcomes.
Adult ; Aged ; COVID-19/virology* ; China/epidemiology* ; Comorbidity ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Severity of Illness Index ; Treatment Outcome

Objective:To investigate the relationship between hypertension with hyperhomocysteinemia (HHcy) and early neurological deterioration (END) in patients with intracerebral hemorrhage.Methods:Patients with acute intracerebral hemorrhage admitted to the Department of Neurology, the First People's Hospital of Huainan from January 2017 to December 2018 were enrolled prospectively. END was defined as the National Institutes of Health Stroke Scale (NIHSS) score increased by ≥4 or Glasgow Coma Scale (GCS) score decreased by ≥2 at any time within 72 h after onset from baseline. The baseline data of the END and non-END groups were compared, and multivariate logistic regression analysis was use to evaluate the independent risk factors for END. Results:A total of 238 patients with acute intracerebral hemorrhage were enrolled, and 64 of them (26.9%) developed END. The baseline hematoma volume, NIHSS score, blood glucose, homocysteine level, neutrophil count, and the proportion of hypertension, hemorrhage into ventricle and hematoma enlargement in the END group were significantly higher than those in the non-END group, while the baseline GCS score was significantly lower than that in the non-END group (all P<0.05). Multivariate logistic regression analysis showed that after adjusting for the confounding factors, the baseline hematoma volume (odds ratio [ OR] 1.086, 95% confidence interval [ CI] 1.029-1.146; P=0.003), baseline GCS score ( OR 0.420, 95% CI 0.245-0.719; P=0.002) and hypertension with HHcy ( OR 2.441, 95% CI 1.185-5.029; P=0.016) had significant independent correlation with END. Conclusion:Hypertension with HHcy is an independent predictor of END in patients with intracerebral hemorrhage.

From November 2014 to July 2017,637 patients with acute coronary syndrome (ACS) were included in the analysis,among whom there were 48 cases with prior ischemic stroke (7.5％).The risk factors,history,severity of coronary artery disease,medication status,and incidence of adverse cardiovascular and cerebrovascular events (cardiac death,re-infarction,heart failure,stroke) were analyzed.Compare with patients without prior ischemic stroke (control group) patients with prior ischemic stroke (study group) had lower rates in administration of beta blockers [50.00％(24/28) vs.69.78％(411/589),x2=8.02,P＜0.05]and interventional therapy[56.67％(17/30) vs.81.86％(334/408),x2=11.15,P＜0.05].However,there were no significant differences in medication of dual antiplatelet,statins and angiotensin converting enzyme inhibitors or angiotensin receptor blocker between two groups (P＞0.05);and there was no significant difference in major adverse cardiovascular events between two groups (P＞0.05).In the future,more studies are needed for clinical management of this group of patients.

Aim To investigate the role and mecha-nism of exchange protein directly activated by cAMP (Epac) protein in the paraventricular nucleus(PVN) of the hypothalamus in the development of inflammatory pain in rats. Methods Adult SD male rats were cho-sen to establish the model of inflammatory pain through subcutaneous injection of complete Freund's adjuvant(CFA) on the center of left hind foot. Western blot was used to detect the changes of the expression of Ep-ac protein. Thermal withdrawal latency(TWL) was ob-served after the PVN injecting 8p-CPT-2′-O-Me-cAMP (8p-CPT),the agonist of Epac. Then activated down-stream MEK1/2 protein of Epac in PVN was detected using Western blot when the potency was the strongest.Results ① Compared with normal saline(control group),TWL decreased significantly on d 1, d 3, d 5, d 7,d 9 on the ipsilateral foot of CFA group rats(P<0.01),whereas it returned to normal level in d 13;the paw mechanical withdrawal threshold(PMWT) de-creased significantly on d 6,d 8,d 10,d 12 and d 14 (P<0.05);②Compared with the control,the Epac1 protein in CFA group rats began to decrease from d 3, and significantly decreased on d 3 and d 9(P<0.05), however the expression of Epac2 had no significant change, meanwhile p-MEK1/2 protein decreased sig-nificantly on d 3(P<0.05);③Compared with micro-injection of saline into the PVN(Saline group), the heat hyperalgesia of 20 min and 1h decreased signifi-cantly and TWL increased significantly after PVN ad-ministration of 8p-CPT(8p-CPT group)(P <0.05);paraventricular nucleus p-MEK1/2 protein expression increased significantly in 30 min(P <0.05) and re-covered to normal level 2 h after administration. Con-clusion The Epac1-MEK1/2 signaling pathway in the paraventricular nucleus of the hypothalamus may be in-volved in the development of chronic inflammatory pain induced by CFA.

Objective:To observe the effect of acupuncture on the expression of mitochondrial proteome in hippocampus of senescence-accelerated mouse prone g (SAMPg) mice models with Alzheimer disease (AD),and to explore the possible protective mechanism of acupuncture on mitochondria.Methods:Sixty 6-month-old male SAMP8 mice were randomly divided into an acupuncture at acupoint group,an acupuncture at non-acupoint group and a model group,20 mice in each group.The 20 male senescence-accelerated mouse/resistance 1 (SAMR1) mice of the same age were used as a normal control group.Shenshu (BL 23),Baihui (GV 20),Xuehai (SP 10) and Geshu (BL 17) were selected for acupuncture intervention in acupuncture at acupoint group.After an 8-week intervention,mitochondrial tissues were extracted from the hippocampus.Differentially expressed proteins were identified by subcellular organelle proteomics.Western blot was used to verify the expressions of some related proteins in hippocampal mitochondria.Results:Compared with the model group,there were 13 differentially expressed protein spots in the acupuncture at acupoint group,of which,9 were up-regulated,including neurofilament light polypeptide (NFL),actin (cytoplasmic 1,database ID:ACTB),tubulin beta-2A chain (TBB2A),tropomodulin-2 (TMOD2),pyruvate dehydrogenase E1 component subunit beta (PDHE1-β),NADH-ubiquinone oxidoreductase 75 kDa subunit (database ID:NDUS1),heat shock cognate 71 kDa protein (HSC71),pyruvate dehydrogenase E1 component subunit alpha (PDHE1-α) and ATP synthase beta subunit (ATP-β);4 were down-regulated,including glial fibrillary acidic protein (GFAP),pyruvate dehydrogenase phosphatase 1 (PDP1),mitochondrial-processing peptidase subunit alpha (MMP-α) and adenosine kinase (ADK).According to the information provided in the protein database,most of the differentially expressed proteins involve the regulation of mitochondrial function and structure.The expression levels of NFL and TBB2A in the normal control group and the acupuncture at acupoint group were significantly higher than those in the acupuncture at non-acupoint group (P＜0.05).ATP-β and NDUS1 expression levels were significantly higher in the acupuncture at acupoint group than those in the acupuncture at non-acupoint group (P＜0.05);there was no significant difference between the acupuncture at non-acupoint group and the model group (P＞0.05).Conclusion:Acupuncture may achieve the potential therapeutic effect on AD by regulating the structure and functional proteins of hippocampal mitochondria.

OBJECTIVETo express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage.

METHODSPCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni-beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of mD1R on the hematopoietic stem cell (HSC), myeloid cells and lymphoid cells by flow cytometry. The LinScal-1c-Kit cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin (IL)-10.

RESULTSThe prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2.96-fold and 6.18-fold. In addition, mD1R improved the amplification of the myeloid progenitor cells and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury.

CONCLUSIONSA new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem cells and hematopoietic microenvironment. The Notch- mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.