Copy number variation encompassing SNP plays an important role in IVD and precision medi-cine.As the most commonly used method,FISH could not overcome the probe cross-reactivity which is common when to detect SNP.Here we developed a quantitative and qualitative method on copy number variation encompassing SNP.In this study,the rs76711854 was used as an example to establish a quanti-tative method by advantage of probe cross-reactivity.The fragment encompassing rs76711854 and its downstream to 9 514 bp were amplified by PCR.The allelic genotypes were verified by Sanger sequen-cing.Different probes with or without cross-reactivity to be used via quantitative real-time PCR and digit-al PCR.The different clusters(2D)and fluorescence intensity layers(1D)exist by adding probe with cross-reactivity.The A/G ratio measured by digital PCR is 2︰1,which is verified by probe targeting to the SNP.The copy-number variant exists in the 9kb-long fragment upstream to the SNP of prostate cancer cell line but not in human endometrial adenocarcinoma cell line Ishikawa.The data suggest that there is a multi-copy variation at this locus in DU145 cells.The method applied here is based on one single cross-reactivity probe via digital PCR.

P53 is a key tumor suppressor gene,which is regulated in many ways.Zinc finger 148(ZNF148)and SP5,as zinc finger transcription factors(TFs),play important roles in tumor suppression and carcinogenesis.The regulatory relationship between these two TFs and p53 has not been reported.In this paper,Ishikawa and A549 cell lines with different p53 expression levels were used as research mod-els to explore the transcriptional regulation of the P53 gene by ZNF148 and SP5.The data showed that there were differences in the expression of ZNF148 and SP5 in the two cell lines.The mRNA expression of ZNF148 in Ishikawa was 1.9 times higher than that of A549,and the mRNA expression of SP5 in A549 was 802.4 times that of ZNF148.Data showed that in Ishikawa cells,the expression of P53 de-creased(81.8％)after ZNF148 knockdown,and increased(2.6 times)after SP5 overexpression.Transfection of si-SP5 and ZNF148 expression plasmids into A549 cells increased the mRNA expression of P53 by 6.6 times and 14.6 times,respectively.These results indicate that ZNF148 could activate,whereas SP5 could inhibit,P53 expression.The conserved cis-element of ZNF148 and SP5 TFs was found in the region of the P53 promoter by bioinformatics methods.The data from dual luciferase reporter gene assay showed that the luciferase activity of ZNF148 in Ishikawa and A549 cells was increased by 2.1-fold and 4.2-fold compared with the control group(P＜0.05).Compared with the control group,the normalized relative luciferase activity of transfected SP5 decreased by 77.1％and 35.7％(P＜0.05).However,when the cis-element of ZNF148 and SP5 was mutated,the effect disappeared.Further trans-fection of ZNF148 and SP5 with different ratios revealed that SP5 could reverse the transcriptional activa-tion of P53 by ZNF148.Studies have shown that ZNF148 shares a common site with SP5,and the ratio of the two TFs may influence the transcriptional activity of P53.The expression of the Wnt pathway and the cell proliferation rate after knockdown of ZNF148 and SP5 were further studied to explore the role of the two TFs.Our data show that ZNF148 and SP5 could regulate the transcriptional activity of P53,and their expression levels and interaction may be the key factors regulating P53 expression.

Copy number variation encompassing SNP plays an important role in IVD and precision medi-cine.As the most commonly used method,FISH could not overcome the probe cross-reactivity which is common when to detect SNP.Here we developed a quantitative and qualitative method on copy number variation encompassing SNP.In this study,the rs76711854 was used as an example to establish a quanti-tative method by advantage of probe cross-reactivity.The fragment encompassing rs76711854 and its downstream to 9 514 bp were amplified by PCR.The allelic genotypes were verified by Sanger sequen-cing.Different probes with or without cross-reactivity to be used via quantitative real-time PCR and digit-al PCR.The different clusters(2D)and fluorescence intensity layers(1D)exist by adding probe with cross-reactivity.The A/G ratio measured by digital PCR is 2︰1,which is verified by probe targeting to the SNP.The copy-number variant exists in the 9kb-long fragment upstream to the SNP of prostate cancer cell line but not in human endometrial adenocarcinoma cell line Ishikawa.The data suggest that there is a multi-copy variation at this locus in DU145 cells.The method applied here is based on one single cross-reactivity probe via digital PCR.

P53 is a key tumor suppressor gene,which is regulated in many ways.Zinc finger 148(ZNF148)and SP5,as zinc finger transcription factors(TFs),play important roles in tumor suppression and carcinogenesis.The regulatory relationship between these two TFs and p53 has not been reported.In this paper,Ishikawa and A549 cell lines with different p53 expression levels were used as research mod-els to explore the transcriptional regulation of the P53 gene by ZNF148 and SP5.The data showed that there were differences in the expression of ZNF148 and SP5 in the two cell lines.The mRNA expression of ZNF148 in Ishikawa was 1.9 times higher than that of A549,and the mRNA expression of SP5 in A549 was 802.4 times that of ZNF148.Data showed that in Ishikawa cells,the expression of P53 de-creased(81.8％)after ZNF148 knockdown,and increased(2.6 times)after SP5 overexpression.Transfection of si-SP5 and ZNF148 expression plasmids into A549 cells increased the mRNA expression of P53 by 6.6 times and 14.6 times,respectively.These results indicate that ZNF148 could activate,whereas SP5 could inhibit,P53 expression.The conserved cis-element of ZNF148 and SP5 TFs was found in the region of the P53 promoter by bioinformatics methods.The data from dual luciferase reporter gene assay showed that the luciferase activity of ZNF148 in Ishikawa and A549 cells was increased by 2.1-fold and 4.2-fold compared with the control group(P＜0.05).Compared with the control group,the normalized relative luciferase activity of transfected SP5 decreased by 77.1％and 35.7％(P＜0.05).However,when the cis-element of ZNF148 and SP5 was mutated,the effect disappeared.Further trans-fection of ZNF148 and SP5 with different ratios revealed that SP5 could reverse the transcriptional activa-tion of P53 by ZNF148.Studies have shown that ZNF148 shares a common site with SP5,and the ratio of the two TFs may influence the transcriptional activity of P53.The expression of the Wnt pathway and the cell proliferation rate after knockdown of ZNF148 and SP5 were further studied to explore the role of the two TFs.Our data show that ZNF148 and SP5 could regulate the transcriptional activity of P53,and their expression levels and interaction may be the key factors regulating P53 expression.

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-β1(TGF-β1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-β1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.
NF-kappa B/metabolism* ; Hepatic Stellate Cells ; Transforming Growth Factor beta1/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Intercellular Adhesion Molecule-1/metabolism* ; Isodon ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Toll-Like Receptor 4/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Lipopolysaccharides/pharmacology* ; Signal Transduction ; Colchicine/pharmacology* ; Caspases

OBJECTIVE: To explore whether acupuncture combined with moxibustion could inhibit epithelialmesenchymal transition in Crohn's disease by affecting the transforming growth factor β 1 (TGF- β 1)/Smad3/Snail pathway. METHODS: Sixty-three patients with Crohn's disease were randomly divided into an observation group (31 cases) receiving moxibustion at 43 °C combined with acupuncture, and a control group (32 cases) receiving moxibustion at 37 °C combined with sham acupuncture using a random number table. Patients were treated for 12 weeks. Crohn's Disease Activity Index (CDAI) was used to evaluate disease activity. Hematoxylin-eosin staining and transmission electron microscopy were utilized to observe the morphological and ultrastructural changes. Immunohistochemistry was used to detect the expression of transforming growth factor β 1 (TGF-β 1), T β R1, T β R2, Smad3, Snail, E-cadherin and fibronectin in intestinal mucosal tissues. RESULTS: The decrease of the CDAI score, morphological and ultrastructural changes were more significant in observation group. The expression levels of TGF- β 1, Tβ R2, Smad3, and Snail in the observation group were significantly lower than those before the treatment (P<0.05 or P<0.01). After treatment, the expression levels of TGF-β 1, TβR2, and Snail in the observation group were significantly lower than those in the control group (all P<0.05); compared with the control group, the expression of fibronectin in the observation group was significantly decreased, and the expression of E-cadherin was significantly increased (all P<0.05). CONCLUSIONS Moxibustion at 43 °C combined with acupuncture may suppress TGF-β 1/Smad3/Snail pathway-mediated epithelial-mesenchymal transition of intestinal epithelial cells in Crohn's disease patients by inhibiting the expression levels of TGF-β 1, Tβ R2, Smad3, and Snail. (Registration No. ChiCTR-IIR-16007751).
Acupuncture Therapy ; Cadherins/metabolism* ; Crohn Disease/therapy* ; Epithelial-Mesenchymal Transition ; Fibronectins/metabolism* ; Humans ; Moxibustion ; Smad3 Protein/metabolism* ; Snail Family Transcription Factors/metabolism* ; Transforming Growth Factor beta1/metabolism*

Pyroptosis， also known as cell inflammatory necrosis， is different from apoptosis， necrosis， and other forms of cell death in morphological characteristics， occurrence， and regulatory mechanism. It is a new type of programmed cell death dependent on Caspase， which has been discovered and confirmed in recent years. Studies have shown that pyroptosis is closely related to the occurrence of liver diseases， and is critical in alcoholic liver disease （ALD）， non-alcoholic fatty liver disease （NAFLD）， liver fibrosis， and liver cancer. Pyroptosis causes inflammatory injury of hepatocytes to promote the occurrence and development of liver diseases by activating Caspase-1， cleaving the effector gasdermin-D （GSDMD）， and releasing pro-inflammatory cytokines， such as interleukin-18 （IL-18） and interleukin-1β （IL-1β） mainly through the classical NOD-like receptor protein 3 （NLRP3） inflammasome pathway. Clinically， Chinese medicine in the treatment of liver diseases has unique efficacy and low side effects. In the intervention on liver diseases， Chinese medicine blocks the pyroptosis signaling pathway to relieve the liver inflammation by inhibiting the assembly and activation of NLRP3 inflammasome multiprotein complex， blunting the activity of Caspase-1 or Caspase-4/Caspase-5/Caspase-11， and inhibiting the cleavage of GSDMD to reduce the release of pro-inflammatory cytokines such as IL-18 and IL-1β. Therefore， in-depth investigation of pyroptosis facilitates unveiling its role in the occurrence， development， and prognosis of liver diseases， and the enhancement or inhibition of pyroptosis may provide a new strategy for the prevention and treatment of liver diseases by Chinese medicine. At present， NLRP3 inflammasome， a key protein in the classic pyroptosis signaling pathway， has become an anti-liver disease target of Chinese medicine. This study briefly summarized the relationship between pyroptosis and liver diseases and reviewed the intervention of monomers， compound prescriptions， effective fractions and extracts of Chinese medicine in recent years to provide important guidance for further exploring the pathogenic mechanism of pyroptosis and the treatment of liver diseases with Chinese medicine.

Blood brain barrier (BBB), as barrier between plasma and brain cells formed by brain capillary wall and glial cells and barrier between plasma and cerebrospinal fluid formed by choroid plexus, can prevent some brain tissues (mostly harmful substances), so as to maintain a stable internal environment of brain tissues, while stopping most drugs from the intracranial and causeing difficulties to cerebral diseases. The establishment of experimental model of BBB is a key technique for drug treatment of craniocerebral diseases. Therefore, the establishment of the BBB model and the study of its permeability change will deepen the understanding of the neuro-vascular interaction and provide an important theoretical basis for the diagnosis and treatment of central nervous system diseases. Traditional Chinese medicine(TCM) can affect brain tissue superoxide dismutase (SOD) activity, inhibit myeloperoxidase (MPO) activity and tumor necrosis factor-α (TNF-α) content, so as to affect the expression of zonula occluden-1 (ZO-1) or up-regulate the expression of Claudin-5, inhibit BBB permeability under pathological conditions, and play a protective role in BBB. TCM can also promote the changes in BBB permeability by affecting the expressions of cell adhesion factor-1 (CAM-1), ZO-1, filamentous actin(F-actin), P-glycoprotein(P-gp)and 5-hydroxytryptamine (5-HT), or increase drug permeability through co-delivery with other drugs. In this paper, the methods, advantages and disadvantages of the establishment of experimental models of the BBB in recent years, as well as the effects of TCM monomers, effective components and TCM compounds on the permeability of the BBB were summarized, so as to provide important guidance and direction for the future treatment of intracranial diseases by traditional Chinese and western medicine.

Liver disease is the general term for all diseases that occur in the liver. In recent years, traditional Chinese medicine has certain advantages in the treatment of liver diseases. With a high experimental study value, good clinical efficacy and less adverse reactions, it has broad prospects. Toll-like receptor 4 (TLR4) signaling pathway is closely related to liver diseases. Its mechanism is to activate nuclear factor-κB (NF-κB) through tlr4-mediated signaling pathway, inhibit the secretion of such inflammatory factors as interleukin-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor-alpha (TNF-α), and the inflammatory damage of liver cells, so as to further inhibit the effect of IL-1,IL-6, TNF-α in activating Hepatic Stellate Cell(HSC). The ways of blocking TRL4 pathway are as follows:Inhibiting the expression of TLR4,Inhibiting the dimerization of TLR4. Blocking intracellular signal transduction:①acting on the binding protein; ②acting on the kinase IRAKs; ③acting on TLRAFst. In these ways, the TRL4 pathway is blocked, the inflammation is inhibited, and the anti-liver disease effect is achieved. Therefore, inhibiting or enhancing TLR4 signaling pathway or intervening in some links of TLR4 signaling pathway has become a new strategy for the treatment of liver diseases. Toll-like receptor 4 signaling pathway has become one of the targets of traditional Chinese medicine (TCM) against liver diseases. In this paper, the recent literatures on the effect of TCM in resisting activation of TLR4 signaling pathway and the effect of anti-liver diseases through monomers and effective parts of TCM, extracts of TCM and compound prescriptions of TCM were collected and summarized to provide important guiding significance and direction for the treatment of liver diseases by TCM and WM in the next step.

In this work,a highly sensitive electrochemical biosensor for the detection of trace adenosine triphosphate (ATP) was proposed.The biosensor was based on porous anodic alumina (PAA) and SiO2 nanoparticles combining with several oligonucleotides to construct sandwich structure.It was characterized by scanning electron microscopy,fluorescence microscopy,differential pulse voltammetry and electrochemical impedance spectroscopy,which conformed to the reliability of the biosensor fabrication and the feasibility of the detection.In the presence of ATP,the sandwich structures could be destroyed.The variation of the current was directly corresponding to the amount of the ATP.The application of SiO2nanoparticles could effectively reduce the background and increase the sensitivity of the biosensor.The calibration curve of ATP was obtained in the range of 0.025-0.900 nmol/L with the detection limit of 13 pmol/L (S/N=3).Also,the biosensor exhibited a good specificity.Besides,the sensor was constructed easily and possessed excellent regeneration ability.The proposed biosensor was applied in detection of real sample such as mice blood.Therefore,the proposed ATP-sensing biosensor could be expected to be applied in clinical,pharmaceutical and environmental detection.