Carious lesions are bacteria-caused destructions of the mineralised dental tissues, marked by the simultaneous activation of immune responses and regenerative events within the soft dental pulp tissue. While major molecular players in tooth decay have been uncovered during the past years, a detailed map of the molecular and cellular landscape of the diseased pulp is still missing. In this study we used single-cell RNA sequencing analysis, supplemented with immunostaining, to generate a comprehensive single-cell atlas of the pulp of carious human teeth. Our data demonstrated modifications in the various cell clusters within the pulp of carious teeth, such as immune cells, mesenchymal stem cells (MSC) and fibroblasts, when compared to the pulp of healthy human teeth. Active immune response in the carious pulp tissue is accompanied by specific changes in the fibroblast and MSC clusters. These changes include the upregulation of genes encoding extracellular matrix (ECM) components, including COL1A1 and Fibronectin (FN1), and the enrichment of the fibroblast cluster with myofibroblasts. The incremental changes in the ECM composition of carious pulp tissues were further confirmed by immunostaining analyses. Assessment of the Fibronectin fibres under mechanical strain conditions showed a significant tension reduction in carious pulp tissues, compared to the healthy ones. The present data demonstrate molecular, cellular and biomechanical alterations in the pulp of human carious teeth, indicative of extensive ECM remodelling, reminiscent of fibrosis observed in other organs. This comprehensive atlas of carious human teeth can facilitate future studies of dental pathologies and enable comparative analyses across diseased organs.
Humans ; Dental Pulp ; Fibronectins ; Extracellular Matrix/pathology* ; Dental Caries ; Sequence Analysis, RNA

This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-β1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-β1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-β1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.
Mice ; Male ; Animals ; Pulmonary Fibrosis/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Epimedium/metabolism* ; Fibronectins/metabolism* ; Matrix Metalloproteinase 7/therapeutic use* ; Matrix Metalloproteinase 8/therapeutic use* ; Vimentin/metabolism* ; Interleukin-6/metabolism* ; Mice, Inbred C57BL ; Lung ; Collagen/metabolism* ; Bleomycin/toxicity* ; RNA, Messenger/metabolism* ; Cadherins/metabolism*

This study explored the effect and mechanism of Maiwei Yangfei Decoction(MWYF) on pulmonary fibrosis(PF) mice. MWYF was prepared, and its main components were detected by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-MS/MS). Male C57BL/6J mice were randomly divided into a control group, a model group, a pirfenidone(PFD) group, and low-, medium-, and high-dose MWYF groups, with 10 mice in each group. The PF model was induced in mice except for those in the control group by intratracheal instillation of bleomycin(BLM), and model mice were treated with saline or MWYF or PFD by gavage the next day. The water consumption, food intake, hair, and activity of mice were observed daily. The pathological changes in lung tissues were observed by hematoxylin-eosin(HE) staining, Masson staining, and CT scanning. The level of hydroxyproline(HYP) in lung tissues was detected by alkaline hydrolysis. Immunohistochemistry was used to observe the expression of collagen type Ⅲ(COL3) and fibronectin. The mRNA expression levels of α-smooth muscle actin(α-SMA), type Ⅰ collagen α1(COL1α1), COL3, and vimentin were detected by reverse transcription real-time fluorescence quantitative polymerase chain reaction(RT-qPCR). Superoxide dismutase(SOD) and malondialdehyde(MDA) kits were used to detect oxidative stress indicators in lung tissues and serum. The nuclear translocation of nuclear factor E2-related factor 2(Nrf2) protein was detected by immunofluorescence. The protein and mRNA expression levels of Nrf2, catalase(CAT), and heme oxygenase 1(HO-1) in lung tissues were detected by Western blot and RT-qPCR. Twelve chemical components were detected by UPLC-MS/MS. Animal experiments showed that MWYF could improve alveolar inflammation, collagen deposition, and fibrosis in PF mice, increase body weight of mice, and down-regulate the expression of fibrosis indexes such as HYP, α-SMA, COL1α1, COL3, fibronectin, and vimentin in lung tissues. In addition, MWYF could potentiate the activity of SOD in lung tissues and serum of PF mice, up-regulate the expression level of Nrf2, and promote its transfer to the nucleus, up-regulate the levels of downstream antioxidant target genes CAT and HO-1, and then reduce the accumulation of lipid metabolite MDA. In summary, MWYF can significantly improve the pathological damage and fibrosis of lung tissues in PF mice, and its mechanism may be related to the activation of the Nrf2 pathway to regulate oxidative stress.
Mice ; Male ; Animals ; Pulmonary Fibrosis/chemically induced* ; NF-E2-Related Factor 2/metabolism* ; Fibronectins/metabolism* ; Vimentin/metabolism* ; Chromatography, Liquid ; Mice, Inbred C57BL ; Tandem Mass Spectrometry ; Oxidative Stress ; Superoxide Dismutase/metabolism* ; RNA, Messenger/metabolism*

OBJECTIVE: To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. METHODS: hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. RESULTS: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. CONCLUSION Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
Pregnancy ; Female ; Humans ; Rabbits ; Animals ; Vascular Endothelial Growth Factor A/metabolism* ; Fibronectins/metabolism* ; Collagen Type I/genetics* ; Tenascin/metabolism* ; Collagen/metabolism* ; Anterior Cruciate Ligament/surgery* ; Mesenchymal Stem Cells ; Tendons/metabolism* ; Fibroblasts/metabolism*

OBJECTIVES: Long-term elevated blood pressure may lead to kidney damage, yet the pathogenesis of hypertensive kidney damage is still unclear. This study aims to explore the role and significance of leucine-rich alpha-2-glycoprotein-1 (LRG-1) in hypertensive renal damage through detecting the levels of LRG-1 in the serum and kidney of mice with hypertensive renal damage and its relationship with related indexes. METHODS: C57BL/6 mice were used in this study and randomly divided into a control group, an angiotensin II (Ang II) group, and an Ang II+irbesartan group. The control group was gavaged with physiological saline. The Ang II group was pumped subcutaneously at a rate of 1.5 mg/(kg·d) for 28 days to establish the hypertensive renal damage model in mice, and then gavaged with equivalent physiological saline. The Ang II+irbesartan group used the same method to establish the hypertensive renal damage model, and then was gavaged with irbesartan. Immunohistochemistry and Western blotting were used to detect the expression of LRG-1 and fibrosis-related indicators (collagen I and fibronectin) in renal tissues. ELISA was used to evaluate the level of serum LRG-1 and inflammatory cytokines in mice. The urinary protein-creatinine ratio and renal function were determined, and correlation analysis was conducted. RESULTS: Compared with the control group, the levels of serum LRG-1, the expression of LRG-1 protein, collagen I, and fibronectin in kidney in the Ang II group were increased (all P<0.01). After treating with irbesartan, renal damage of hypertensive mice was alleviated, while the levels of LRG-1 in serum and kidney were decreased, and the expression of collagen I and fibronectin was down-regulated (all P<0.01). Correlation analysis showed that the level of serum LRG-1 was positively correlated with urinary protein-creatinine ratio, blood urea nitrogen, and blood creatinine level in hypertensive kidney damage mice. Serum level of LRG-1 was also positively correlated with serum inflammatory factors including TNF-α, IL-1β, and IL-6. CONCLUSIONS Hypertensive renal damage mice display elevated expression of LRG-1 in serum and kidney, and irbesartan can reduce the expression of LRG-1 while alleviating renal damage. The level of serum LRG-1 is positively correlated with the degree of hypertensive renal damage, suggesting that it may participate in the occurrence and development of hypertensive renal damage.
Animals ; Mice ; Mice, Inbred C57BL ; Fibronectins ; Irbesartan ; Creatinine ; Kidney/physiology* ; Hypertension/complications* ; Angiotensin II ; Collagen Type I

Ivermectin is a US Food and Drug Administration (FDA)-approved antiparasitic agent with antiviral and anti-inflammatory properties. Although recent studies reported the possible anti-inflammatory activity of ivermectin in respiratory injuries, its potential therapeutic effect on pulmonary fibrosis (PF) has not been investigated. This study aimed to explore the ability of ivermectin (0.6 mg/kg) to alleviate bleomycin-induced biochemical derangements and histological changes in an experimental PF rat model. This can provide the means to validate the clinical utility of ivermectin as a treatment option for idiopathic PF. The results showed that ivermectin mitigated the bleomycin-evoked pulmonary injury, as manifested by the reduced infiltration of inflammatory cells, as well as decreased the inflammation and fibrosis scores. Intriguingly, ivermectin decreased collagen fiber deposition and suppressed transforming growth factor-‍β1 (TGF-‍β1) and fibronectin protein expression, highlighting its anti-fibrotic activity. This study revealed for the first time that ivermectin can suppress the nucleotide-binding oligomerization domain (NOD)‍-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, as manifested by the reduced gene expression of NLRP3 and the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), with a subsequent decline in the interleukin‍-‍1β (IL‍-‍1β) level. In addition, ivermectin inhibited the expression of intracellular nuclear factor-‍κB (NF‍-‍κB) and hypoxia‑inducible factor‑1α (HIF‍-‍1α) proteins along with lowering the oxidative stress and apoptotic markers. Altogether, this study revealed that ivermectin could ameliorate pulmonary inflammation and fibrosis induced by bleomycin. These beneficial effects were mediated, at least partly, via the downregulation of TGF-‍β1 and fibronectin, as well as the suppression of NLRP3 inflammasome through modulating the expression of HIF‑1α and NF-‍κB.
Animals ; Rats ; Anti-Inflammatory Agents ; Bleomycin/toxicity* ; Fibronectins/metabolism* ; Fibrosis ; Inflammasomes/metabolism* ; Ivermectin/adverse effects* ; NF-kappa B/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pulmonary Fibrosis/drug therapy*

Objective: To investigate the mechanism of S100A7 inducing the migration and invasion in cervical cancers. Methods: Tissue samples of 5 cases of cervical squamous cell carcinoma and 3 cases of adenocarcinoma were collected from May 2007 to December 2007 in the Department of Gynecology of the Affiliated Hospital of Qingdao University. Immunohistochemistry was performed to evaluate the expression of S100A7 in cervical carcinoma tissues. S100A7-overexpressing HeLa and C33A cells were established with lentiviral systems as the experimental group. Immunofluorescence assay was performed to observe the cell morphology. Transwell assay was taken to detect the effect of S100A7-overexpression on the migration and invasion of cervical cancer cells. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to examine the mRNA expressions of E-cadherin, N-cadherin, vimentin and fibronectin. The expression of extracellular S100A7 in conditioned medium of cervical cancer cell was detected by western blot. Conditioned medium was added into Transwell lower compartment to detect cell motility. Exosomes were isolated and extracted from the culture supernatant of cervical cancer cell, the expressions of S100A7, CD81 and TSG101 were detected by western blot. Transwell assay was taken to detect the effect of exosomes on the migration and invasion of cervical cancer cells. Results: S100A7 expression was positively expressed in cervical squamous carcinoma and negative expression in adenocarcinoma. Stable S100A7-overexpressing HeLa and C33A cells were successfully constructed. C33A cells in the experimental group were spindle shaped while those in the control group tended to be polygonal epithelioid cells. The number of S100A7-overexpressed HeLa cells passing through the Transwell membrane assay was increased significantly in migration and invasion assay (152.00±39.22 vs 105.13±15.75, P<0.05; 115.38±34.57 vs 79.50±13.68, P<0.05). RT-qPCR indicated that the mRNA expressions of E-cadherin in S100A7-overexpressed HeLa and C33A cells decreased (P<0.05) while the mRNA expressions of N-cadherin and fibronectin in HeLa cells and fibronectin in C33A cells increased (P<0.05). Western blot showed that extracellular S100A7 was detected in culture supernatant of cervical cancer cells. HeLa cells of the experimental group passing through transwell membrane in migration and invasion assays were increased significantly (192.60±24.41 vs 98.80±47.24, P<0.05; 105.40±27.38 vs 84.50±13.51, P<0.05) when the conditional medium was added into the lower compartment of Transwell. Exosomes from C33A cell culture supernatant were extracted successfully, and S100A7 expression was positive. The number of transmembrane C33A cells incubated with exosomes extracted from cells of the experimental group was increased significantly (251.00±49.82 vs 143.00±30.85, P<0.05; 524.60±52.74 vs 389.00±63.23, P<0.05). Conclusion: S100A7 may promote the migration and invasion of cervical cancer cells by epithelial-mesenchymal transition and exosome secretion.
Female ; Humans ; Uterine Cervical Neoplasms/pathology* ; HeLa Cells ; Fibronectins/metabolism* ; Culture Media, Conditioned ; Carcinoma, Squamous Cell/metabolism* ; Adenocarcinoma ; Cadherins/metabolism* ; RNA, Messenger/metabolism* ; Cell Movement ; Epithelial-Mesenchymal Transition/genetics* ; Cell Line, Tumor ; Cell Proliferation ; S100 Calcium Binding Protein A7/metabolism*

Humans ; Glomerulonephritis, Membranoproliferative/pathology* ; Fibronectins

Objective: To investigate the effects of low-dose photodynamic therapy on the proliferation, regulation, and secretion functions of human adipose mesenchymal stem cells (ADSCs) and the related mechanism, so as to explore a new method for the repair of chronic wounds. Methods: The experimental research methods were adopted. From February to April 2021, 10 patients (5 males and 5 females, aged 23 to 47 years) who underwent cutaneous surgery in the Department of Dermatology of the First Affiliated Hospital of Army Medical University (the Third Military Medical University) donated postoperative waste adipose tissue. The cells were extracted from the adipose tissue and the phenotype was identified. Three batches of ADSCs were taken, with each batch of cells being divided into normal control group with conventional culture only, photosensitizer alone group with conventional culture after being treated with Hemoporfin, irradiation alone group with conventional culture after being treated with red light irradiation, and photosensitizer+irradiation group with conventional culture after being treated with Hemoporfin and red light irradiation, with sample number of 3 in each group. At culture hour of 24 after the treatment of the first and second batches of cells, the ADSC proliferation level was evaluated by 5-ethynyl-2'-deoxyuridine staining method and the migration percentage of HaCaT cells cocultured with ADSCs was detected by Transwell experiment, respectively. On culture day of 7 after the treatment of the third batch of cells, the extracellular matrix protein expression of ADSCs was detected by immunofluorescence method. The ADSCs were divided into 0 min post-photodynamic therapy group, 15 min post-photodynamic therapy group, 30 min post-photodynamic therapy group, and 60 min post-photodynamic therapy group, with 3 wells in each group. Western blotting was used to detect the protein expressions and calculate the phosphorylated mammalian target of rapamycin complex (p-mTOR)/mammalian target of rapamycin (mTOR), phosphorylated p70 ribosomal protein S6 kinase (p-p70 S6K)/p70 ribosomal protein S6 kinase (p70 S6K) ratio at the corresponding time points after photodynamic therapy. Two batches of ADSCs were taken, and each batch was divided into normal control group, photodynamic therapy alone group, and photodynamic therapy+rapamycin group, with 3 wells in each group. At culture minute of 15 after the treatment, p-mTOR/mTOR and p-p70 S6K/p70 S6K ratios of cells from the first batch were calculated and detected as before. On culture day of 7 after the treatment, extracellular matrix protein expression of cells from the second batch was detected as before. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: After 12 d of culture, the cells were verified as ADSCs. At culture hour of 24 after the treatment, the ADSC proliferation level ((4.0±1.0)% and (4.1±0.4)%, respectively) and HaCaT cell migration percentages (1.17±0.14 and 1.13±0.12, respectively) in photosensitizer alone group and irradiation alone group were similar to those of normal control group ((3.7±0.6)% and 1.00±0.16, respectively, P>0.05), and were significantly lower than those of photosensitizer+irradiation group ((34.2±7.0)% and 2.55±0.13, respectively, P<0.01). On culture day of 7 after the treatment, compared with those in normal control group, the expression of collagen Ⅲ in ADSCs of photosensitizer alone group was significantly increased (P<0.05), and the expressions of collagen Ⅰ and collagen Ⅲ in ADSCs of irradiation alone group were significantly increased (P<0.01). Compared with those in photosensitizer alone group and irradiation alone group, the expressions of collagen Ⅰ, collagen Ⅲ, and fibronectin of ADSCs in photosensitizer+irradiation group were significantly increased (P<0.01). Compared with those in 0 min post-photodynamic therapy group, the ratios of p-mTOR/mTOR and p-p70 S6K/p70 S6K of ADSCs in 15 min post-photodynamic therapy group were significantly increased (P<0.01), the ratios of p-p70 S6K/p70 S6K of ADSCs in 30 min post-photodynamic therapy group and 60 min post-photodynamic therapy group were both significantly increased (P<0.01). At culture minute of 15 after the treatment, compared with those in normal control group, the ratios of p-mTOR/mTOR and p-p70 S6K/p70 S6K of ADSCs in photodynamic therapy alone group were significantly increased (P<0.05 or P<0.01). Compared with those in photodynamic therapy alone group, the ratios of p-mTOR/mTOR and p-p70 S6K/p70 S6K of ADSCs in photodynamic therapy+rapamycin group were significantly decreased (P<0.05). On culture day of 7 after the treatment, compared with those in normal control group, the expressions of collagen Ⅰ, collagen Ⅲ, and fibronectin of ADSCs in photodynamic therapy alone group were significantly increased (P<0.01). Compared with those in photodynamic therapy alone group, the expressions of collagen Ⅰ, collagen Ⅲ, and fibronectin of ADSCs in photodynamic therapy+rapamycin group were significantly decreased (P<0.01). Conclusions: Low-dose photodynamic therapy can promote the proliferation of ADSCs, improve the ability of ADSCs to regulate the migration of HaCaT cells, and enhance the secretion of extracellular matrix protein by rapidly activating mTOR signaling pathway.
Adipose Tissue ; Female ; Fibronectins ; Humans ; Male ; Mesenchymal Stem Cells ; Photochemotherapy ; Photosensitizing Agents/pharmacology* ; Sirolimus/pharmacology* ; TOR Serine-Threonine Kinases

The purpose of this study was to investigate the effect of Geniposide on hepatic fibrosis and activation of hepatic stellate cells (HSCs) and to explore possible underlying mechanism. Human HSCs (LX-2) were treated with 5 ng/mL transforming growth factor-β1 (TGF-β1), followed by co-culture with Geniposide at various concentrations (0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100 μmol/L). Cell viability was determined by MTT assay. Then, LX-2 cells were divided into control, TGF-β1 (5 ng/mL) and TGF-β1 + Geniposide (20 μmol/L) groups, and the gene and protein expression of collagen I, fibronectin, α-smooth muscle actin (α-SMA), p-Smad2 and p-Smad3 was detected by qPCR and Western blot, respectively. BALB/c mice were treated with CCl₄ (25%, 1 mL/kg) to generate a model of hepatic fibrosis (CCl₄ group), and the control group and CCl₄ + Geniposide group were administered with olive oil and CCl₄ + 40 mg/kg Geniposide, respectively. After 4 weeks of treatment, the liver function and serum hepatic fibrosis indexes of mice were detected, histological observation was performed by HE and Masson staining, and α-SMA expression in the tissue was analyzed by immunohistochemistry. Western blot was utilized for the determination of the protein expression of α-SMA, TGF-β1, p-Smad2 and p-Smad3. The results showed that Geniposide inhibited LX-2 cell proliferation. In addition, Geniposide significantly downregulated the gene and protein expression of collagen I, fibronectin and α-SMA and the expression of TGF-β1/Smad signaling-related proteins induced by TGF-β1 in vitro. Histological observations showed that Geniposide significantly inhibited CCl₄-induced hepatic fibrosis, HSC activation and expression of TGF-β1/Smad signaling-related proteins in mice. In summary, Geniposide prevents the hepatic fibrosis and HSC activation possibly through the inhibition of the TGF-β1/Smad signaling pathway.
Animals ; Collagen Type I/metabolism* ; Fibronectins ; Hepatic Stellate Cells/pathology* ; Iridoids ; Liver Cirrhosis/pathology* ; Mice ; Signal Transduction ; Smad Proteins/pharmacology* ; Transforming Growth Factor beta1/metabolism*