Objective To explore the mechanism by which gentiopicroside (GPS) prevents macrophage-mediated hepatic fibrosis by regulating the macrophage migration inhibitory factor (MIF)-secreted phosphoprotein 1 (SPP1) signaling pathway in hepatic stellate cells. Methods LX-2 cells were divided into control group, transforming growth factor β(TGF-β) group, and TGF-β combined with GPS (25, 50, 100, 150 μmol/mL) groups. Cell proliferation was detected by EDU assay, cell invasion was assessed by Transwell^TM assay, and the protein expressions of α-smooth muscle actin (α-SMA) and type I collagen (COL1A1) were measured by Western blot. M1-type macrophage-conditioned medium (M1-CM) was used to treat LX-2 cells in the TGF-β group and TGF-β combined with GPS group. The concentrations of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) in the cell supernatant, as well as cell proliferation, invasion ability, and the expressions of α-SMA and COL1A1 were detected. Bioinformatics analysis was performed to identify the target intersections of GPS, hepatic fibrosis, and macrophage-related genes. Drug affinity responsive target stability (DARTS) experiments and Western blot were used to verify the regulatory effect of GPS on MIF. Furthermore, LX-2 cells were divided into control group, TGF-β group, TGF-β combined with M2-CM group, TGF-β and oe-NC combined with M2-CM group, and TGF-β and oe-MIF combined with M2-CM group to analyze the concentrations of iNOS and Arg1 in the cell supernatant, as well as changes in cell proliferation, invasion, and the expressions of α-SMA and COL1A1. LX-2 cells were also divided into control group, TGF-β group, TGF-β combined with oe-NC group, TGF-β combined with oe-MIF group, and TGF-β and oe-MIF combined with GPS group to determine the protein expressions of MIF and SPP1 by Western blot. A rat model of hepatic fibrosis was constructed to explore the potential therapeutic effects of GPS on hepatic fibrosis in vivo. Results Compared with the control group, the proliferation and invasion abilities of LX-2 cells in the TGF-β group were increased, and the protein expressions of α-SMA and COL1A1 were enhanced. GPS intervention inhibited the proliferation and invasion of LX-2 cells under TGF-β conditions and reduced the expressions of α-SMA and COL1A1. Compared with the control group, the concentration of iNOS in the cell supernatant of the TGF-β group was upregulated, while the concentration of Arg1 was decreased. M1-CM treatment further increased the concentration of iNOS, decreased the concentration of Arg1, and promoted cell proliferation and invasion, as well as upregulated the expressions of α-SMA and COL1A1 on the basis of TGF-β intervention. However, GPS could reverse the effects of M1-CM intervention. Bioinformatics analysis revealed that MIF was one of the target intersections of GPS, hepatic fibrosis, and macrophage-related genes, and GPS could target and inhibit its expression. Compared with the TGF-β group, after M2-CM intervention, the concentration of iNOS in the cell supernatant decreased, the concentration of Arg1 increased, the proliferation and invasion abilities of LX-2 cells were reduced, and the expressions of α-SMA and COL1A1 were weakened. However, overexpression of MIF reversed the effects of M2-CM intervention. Western blot results showed that compared with the control group, the protein expressions of MIF and SPP1 were enhanced in the TGF-β group. Overexpression of MIF further enhanced the expressions of MIF and SPP1, while GPS intervention inhibited the expressions of MIF and SPP1. In the animal experiment, GPS intervention treatment alleviated liver injury in rats with hepatic fibrosis and inhibited the expressions of MIF and SPP1, as well as α-SMA and COL1A1 in liver tissue. Conclusion GPS may prevent macrophage-mediated hepatic fibrosis by inhibiting the MIF-SPP1 signaling pathway in hepatic stellate cells.
Hepatic Stellate Cells/metabolism* ; Signal Transduction/drug effects* ; Macrophage Migration-Inhibitory Factors/genetics* ; Liver Cirrhosis/prevention & control* ; Macrophages/drug effects* ; Iridoid Glucosides/pharmacology* ; Humans ; Cell Proliferation/drug effects* ; Animals ; Cell Line ; Collagen Type I/metabolism* ; Collagen Type I, alpha 1 Chain ; Intramolecular Oxidoreductases/genetics* ; Rats ; Transforming Growth Factor beta/pharmacology* ; Actins/metabolism*

Stem cell treatment may enhance erectile dysfunction (ED) in individuals with cavernous nerve injury (CNI). Nevertheless, no investigations have directly ascertained the implications of varying amounts of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) on ED. We compare the efficacy of three various doses of HUC-MSCs as a therapeutic strategy for ED. Sprague-Dawley rats (total = 175) were randomly allocated into five groups. A total of 35 rats underwent sham surgery and 140 rats endured bilateral CNI and were treated with vehicles or doses of HUC-MSCs (1 × 10 6 cells, 5 × 10 6 cells, and 1 × 10 7 cells in 0.1 ml, respectively). Penile tissues were harvested for histological analysis on 1 day, 3 days, 7 days, 14 days, 28 days, 60 days, and 90 days postsurgery. It was found that varying dosages of HUC-MSCs enhanced the erectile function of rats with bilateral CNI and ED. Moreover, there was no significant disparity in the effectiveness of various dosages of HUC-MSCs. However, the expression of endothelial markers (rat endothelial cell antigen-1 [RECA-1] and endothelial nitric oxide synthase [eNOS]), smooth muscle markers (alpha smooth muscle actin [α-SMA] and desmin), and neural markers (neurofilament [RECA-1] and neurogenic nitric oxide synthase [nNOS]) increased significantly with prolonged treatment time. Masson's staining demonstrated an increased in the smooth muscle cell (SMC)/collagen ratio. Significant changes were detected in the microstructures of various types of cells. In vivo imaging system (IVIS) analysis showed that at the 1 st day, the HUC-MSCs implanted moved to the site of damage. Additionally, the oxidative stress levels were dramatically reduced in the penises of rats administered with HUC-MSCs.
Male ; Animals ; Erectile Dysfunction/metabolism* ; Rats, Sprague-Dawley ; Mesenchymal Stem Cell Transplantation/methods* ; Rats ; Penis/pathology* ; Humans ; Disease Models, Animal ; Umbilical Cord/cytology* ; Peripheral Nerve Injuries/complications* ; Mesenchymal Stem Cells ; Nitric Oxide Synthase Type III/metabolism* ; Actins/metabolism* ; Nitric Oxide Synthase Type I/metabolism*

Five previously undescribed diterpenoids, named succipenoids D‒H (1‒5), along with four undescribed lignans, named succignans A‒D (6‒9), were isolated from the dichloromethane extract of Chinese medicinal succinum. Compounds 1‒5 were characterized as nor-abietane diterpenoids, while compounds 6‒9 were identified as lignans polymerized from two groups of phenylpropanoid units. The structures of these novel compounds, including their absolute configurations, were determined through spectroscopic and computational methods. Biological assessments of renal fibrosis demonstrated that compounds 6 and 7 effectively reduce the expression of proteins associated with renal fibrosis, including α-smooth muscle actin (α-SMA), collagen I, and fibronectin in transforming growth factor-β1 (TGF-β1) induced normal rat kidney proximal tubular epithelial cells (NRK-52e).
Animals ; Rats ; Lignans/isolation & purification* ; Diterpenes/isolation & purification* ; Fibrosis/drug therapy* ; Drugs, Chinese Herbal/pharmacology* ; Molecular Structure ; Cell Line ; Kidney Diseases/pathology* ; Transforming Growth Factor beta1/genetics* ; Kidney/metabolism* ; Actins/genetics* ; Fibronectins/genetics* ; Collagen Type I/genetics* ; Epithelial Cells/metabolism*

Objective To investigate the effect of interleukin-6 (IL-6) on the phagocytosis of MH-S alveolar macrophages and its related mechanisms. Methods A mouse acute lung injury (ALI) model was constructed by instilling lipopolysaccharide (LPS) into the airway. ELISA was used to detect the content of IL-6 in bronchoalveolar lavage fluid (BALF). In vitro cultured MH-S cells, in the presence or absence of signal transducer and activator 3 of transcription(STAT3) inhibitor Stattic (5 μmol/L), IL-6 (10 ng/mL~500 ng/mL) was added to stimulate for 6 hours, and then incubated with fluorescent microspheres for 2 hours. The phagocytosis of MH-S cells was detected by flow cytometry. Western blot analysis was used to detect the expression levels of phosphorylated Janus kinase 2 (p-JAK2), phosphorylated STAT3 (p-STAT3), actin-related protein 2 (Arp2) and filamentous actin (F-actin). Results The content of IL-6 in BALF was significantly increased after the mice were injected with LPS through the airway. With the increase of IL-6 stimulation concentration, the phagocytic function of MH-S cells was enhanced, and the expression levels of Arp2 and F-actin proteins in MH-S cells were increased. The expression levels of p-JAK2 and p-STAT3 proteins increased in MH-S cells stimulated with IL-6(100 ng/mL). After blocking STAT3 signaling, the effect of IL-6 in promoting phagocytosis of MH-S cells disappeared completely, and the increased expression of Arp2 and F-actin proteins in MH-S cells induced by IL-6 was also inhibited. Conclusion IL-6 promotes the expression of Arp2 and F-actin proteins by activating the JAK2/STAT3 signaling pathway, thereby enhancing the phagocytic function of MH-S cells.
Animals ; Mice ; Actins ; Disease Models, Animal ; Interleukin-6 ; Janus Kinase 2 ; Lipopolysaccharides ; Macrophages, Alveolar ; Signal Transduction

OBJECTIVE: To investigate the anti-adhesive effect and underlying mechanism of dynamic and static stress stimulation on the early healing process of rat Achilles tendon injury. METHODS: Achilles tendon tissues of 15 male Sprague Dawley (SD) rats aged 4-6 weeks were isolated and cultured by enzyme digestion method. Rat Achilles tendon cells were treated with tumor necrosis factor α to construct the Achilles tendon injury cell model, and dynamic stress stimulation (dynamic group) and static stress stimulation (static group) were applied respectively, while the control group was not treated. Live/dead cell double staining was used to detect cell activity, ELISA assay was used to detect the expression of α smooth muscle actin (α-SMA), and real-time fluorescence quantitative PCR was used to detect the mRNA expression of collagen type Ⅰ (COL1A1), collagen type Ⅲ (COL3A1), and Scleraxis (SCX). Thirty male SD rats aged 4-6 weeks underwent Achilles tendon suture and were randomly divided into dynamic group (treated by dynamic stress stimulation), static group (treated by static stress stimulation), and control group (untreated), with 10 rats in each group. HE staining and scoring were performed to evaluate the healing of Achilles tendon at 8 days after operation. COL1A1 and COL3A1 protein expressions were detected by immunohistochemical staining, α-SMA and SCX protein expressions were detected by Western blot, and maximum tendon breaking force and tendon stiffness were detected by biomechanical stretching test. RESULTS: In vitro cell experiment, when compared to the static group, the number of living cells in the dynamic group was higher, the expression of α-SMA protein was decreased, the relative expression of COL3A1 mRNA was decreased, and the relative expression of SCX mRNA was increased, and the differences were all significant ( P<0.05). In the in vivo animal experiment, when compared to the static group, the tendon healing in the dynamic group was better, the HE staining score was lower, the expression of COL1A1 protein was increased, the expression of COL3A1 protein was decreased, the relative expression of SCX protein was increased, the relative expression of α-SMA protein was decreased, and the tendon stiffness was increased, the differences were all significant ( P<0.05). CONCLUSION Compared with static stress stimulation, the dynamic stress stimulation improves the fibrosis of the scar tissue of the rat Achilles tendon, promote the recovery of the biomechanical property of the Achilles tendon, and has obvious anti-adhesion effect.
Animals ; Achilles Tendon/injuries* ; Male ; Rats ; Rats, Sprague-Dawley ; Collagen Type I/metabolism* ; Collagen Type III/metabolism* ; Tendon Injuries/therapy* ; Wound Healing ; Stress, Mechanical ; Actins/metabolism* ; Cells, Cultured ; Tissue Adhesions/prevention & control* ; Tumor Necrosis Factor-alpha/metabolism* ; RNA, Messenger/genetics* ; Disease Models, Animal ; Collagen Type I, alpha 1 Chain/metabolism* ; Biomechanical Phenomena ; Basic Helix-Loop-Helix Transcription Factors

OBJECTIVES: To investigate the inhibitory effect of Danshen Injection on endothelial-mesenchymal transition (EndMT) induced by peritoneal dialysis fluid in HMrSV5 cells and the role of the TGF‑β/Smad signaling pathway in mediating this effect. METHODS: HMrSV5 cells cultured in 40% peritoneal dialysis solution for 72 h to induce EndMT were treated with 0.05%, 0.1% and 0.5% Danshen Injection. CCK-8 assay was used to assess the changes in viability of the treated cells, and the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF) in the cell supernatant were detected using ELISA; Western blotting was performed to detect the protein expressions of E-cadherin, α-smooth muscle actin (α-SMA), p-Smad 2/3, and Smad 7 in the cells. RESULTS: Culture in 40% peritoneal dialysis fluid for 72 induced significant EndMT in HMrSV5 cells, which exhibited obviously lowered cell viability. Danshen Injection within the concentration range of 0.025%-1.5% did not significantly affect the viability of the cells. Exposure of HMrSV5 cells to peritoneal dialysis fluid for 72 h significantly increased the production of IL-6, TNF‑α, TGF‑β and VEGF, upregulated the protein expressions of α‑SMA and p-Smad 2/3, and lowered the expressions of E-cadherin and Smad7 proteins. Treatment of the exposed cells with Danshen injection significantly increased cell viability and cellular expressions of E-cadherin and Smad 7 proteins and reduced the production of IL-6, TNF-α, TGF-β and VEGF and the protein expressions of α‑SMA and p-Smad 2/3. CONCLUSIONS Danshen Injection can suppress peritoneal dialysis fluid-induced EndMT in HMrSV5 cells possibly by regulating the TGF-β/Smad signaling pathway.
Signal Transduction/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Transforming Growth Factor beta/metabolism* ; Humans ; Peritoneal Dialysis/adverse effects* ; Salvia miltiorrhiza ; Epithelial-Mesenchymal Transition/drug effects* ; Smad Proteins/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Cell Line ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Cadherins/metabolism* ; Actins/metabolism* ; Dialysis Solutions ; Endothelial-Mesenchymal Transition

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.
Male ; Mice ; Animals ; Keratin-18 ; Actins ; Desmin ; Liver ; Hepatocytes ; Hepatic Stellate Cells

OBJECTIVES: To analyze the clinicopathological features of maxillofacial granular cell tumors (GCT) with the aid of immunohistochemical staining. METHODS: Seven cases of maxillofacial GCT were retrospectively collated, and the microscopic morphology of maxillofacial GCT was analyzed. The expression of S-100, neuron-specific enolase (NSE), SOX-10, CD68, actin, desmin, and Ki-67 in GCT was detected by immunohistochemical staining. The cases were observed in the follow-ups after clinical treatment. RESULTS: All seven GCT tumors lacked envelopes and were poorly defined. Microscopically, the sizes of the tumor cells were large and appeared with inconspicuous cell membranes, forming a syncytium-like appearance. The cytoplasm was filled with characteristic eosinophilic granules. The immunohistochemical results showed that six cases were NSE-positive, five cases were S-100-positive, seven cases were CD68-positive, five cases were SOX-10-positive, one case was actin-positive, and seven cases were desmin-negative. The Ki-67 index did not exceed 5% in all cases. In the follow-up sessions, none of the six cases presented a recurrence. CONCLUSIONS Maxillofacial GCT has a characteristic histological structure. Immunohistochemical S-100, CD68, and other indicators can assist in diagnosis, and the prognosis is good after clinical resection.
Humans ; Ki-67 Antigen/metabolism* ; Granular Cell Tumor/surgery* ; Retrospective Studies ; Actins/metabolism* ; Desmin/metabolism* ; S100 Proteins/metabolism*

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.
Real-Time Polymerase Chain Reaction/methods* ; Paeonia/genetics* ; Actins/genetics* ; Reproducibility of Results ; Transcriptome ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Reference Standards ; Gene Expression Profiling/methods*

Epilepsy is a common, chronic neurological disorder that has been associated with impaired neurodevelopment and immunity. The chemokine receptor CXCR5 is involved in seizures via an unknown mechanism. Here, we first determined the expression pattern and distribution of the CXCR5 gene in the mouse brain during different stages of development and the brain tissue of patients with epilepsy. Subsequently, we found that the knockdown of CXCR5 increased the susceptibility of mice to pentylenetetrazol- and kainic acid-induced seizures, whereas CXCR5 overexpression had the opposite effect. CXCR5 knockdown in mouse embryos via viral vector electrotransfer negatively influenced the motility and multipolar-to-bipolar transition of migratory neurons. Using a human-derived induced an in vitro multipotential stem cell neurodevelopmental model, we determined that CXCR5 regulates neuronal migration and polarization by stabilizing the actin cytoskeleton during various stages of neurodevelopment. Electrophysiological experiments demonstrated that the knockdown of CXCR5 induced neuronal hyperexcitability, resulting in an increased number of seizures. Finally, our results suggested that CXCR5 deficiency triggers seizure-related electrical activity through a previously unknown mechanism, namely, the disruption of neuronal polarity.
Animals ; Humans ; Mice ; Actin Cytoskeleton/metabolism* ; Actins/metabolism* ; Epilepsy/metabolism* ; Neurons/metabolism* ; Receptors, CXCR5/metabolism* ; Seizures/metabolism*