ABSTRACT
OBJECTIVE@#To investigate the effect of Bushen Chushi decoction combined with platelet-rich plasma(PRP) to treat knee osteoarthritis(KOA) in early and middle stage and its regulation on TGF-β1 and Smad-1 expression in serum.@*METHODS@#Total of 45 patients with KOA in early and middle stage from May 2020 to April 2022 were treated and divided into control group and observation group. In control group, there were 30 patients including 12 males and 18 females, aged from 43 to 69 years old with an average of(57.3±6.5) years old and disease duration ranged from 1.5 to 5.0 years with an average of(3.8±1.7) years, and there were 8 cases in gradeⅠ, 13 cases in gradeⅡ, and 9 cases in grade Ⅲ according to Kellgren-Lawrence Grade, PRP 5 ml was injected into knee joint on the first day of No1, 3 week together for 2 times. In the observation group, there were 15 cases including 7 males and 8 females, aged from 45 to 70 years old with an average of (56.7±6.2) years old and disease duration ranged from 1.8 to 5.7 years with an average of (4.0±1.8) years, there were 4 cases in gradeⅠ, 9 cases in gradeⅡand 4 cases in grade Ⅲ according to the Kellgren-Lawrence Grade, PRP 5 ml were injected into knee joints that the time and frequency were the same as those in the control group, and at the same time Bushen Chushi decoction orally were taken 1 dose per day with a total of 28 doses. All patients were treated for four weeks. Visual analogue scale(VAS) and Lequesne MG score before and after treatment were used to evaluate improvement of knee pain and joint function. The TGF-β1 and Smad-1 levels in serum were measured before and after treatment in two groups. The incidence of complications in two groups was observed.@*RESULTS@#All patients were followed up for 26 to 30 days with an average of (28.0±0.6) days. There was no significant difference in VAS and knee Lequesne MG scores between two groups before treatment(P>0.05). The scores of VAS and knee Lequesne MG on the first day after treatment in both groups were lower than those before treatment(P<0.05). The VAS and knee Lequesne MG scores in observation group were lower than those in control group(P<0.05) on the first day after treatment. The TGF-β1 level in serum after treatment were higher significantly than that before treatment in two groups(P<0.05). After treatment, TGF-β1 level in serum in observation group were lower than those in control group with statistically significant differences(P<0.05). The Smad-1 levels in serum after treatment in observation group were higher significantly than that in control group(P<0.05). The levels of Smad-1 were not statistically significant between before and after treatment(P>0.05). There was no significant difference in postopertaive complications between two groups (P>0.05).@*CONCLUSION@#The efficacy of Bushen Chushi decoction combined with PRP in treatment of early and middle KOA is better than that of PRP injection alone. The combined treatment could reduce TGF-β1 level and increase Smad-1 level in serum, which may be a mechanism to inhibit inflammation and alleviate cartilage degeneration to some extent.
ABSTRACT
BACKGROUND: Axons do not regenerate after central nervous system injury in mammals. It is mainly caused by the inhibitory microenvironment at the site of damage and the weakened self-regeneration ability. Studies have found that peripheral nervous system has certain regeneration ability after injury, so we explore the methods of central nervous system repair by studying the genes promoting peripheral nervous system regeneration. As one of the important protein kinase families of neurons, CaMKII up-regulation can improve the ability of neuron regeneration. Similarly, acute depletion of the Smad1 protein in adult mice also prevented axon regeneration in vivo. These genes can directly or indirectly regulate neuronal axon regeneration, but exactly how they regulate neuronal regeneration is still unclear. OBJECTIVE: To study the effects of CaMKII-Smad1 signaling pathway on axon regeneration of dorsal root ganglion neurons by intraperitoneal injection of CaMKII inhibitor and activator, and explored the mechanism of CaMKII and Smad1 in regulating axon regeneration of dorsal root ganglion neurons. METHODS: Totally 40 ICR mice were randomly divided into four groups: KN93 control group, KN93 experimental group, CdCl2 control group and CdCl2 experimental group. Dorsal root ganglion tissue was taken for in vitro culture after 7 days of continuous administration of CaMKII inhibitor KN93 and activator CdCl2. The length of axonal regeneration of dorsal root ganglion neurons was statistically analyzed after 3 days. Protein expression of p-Smad1 in dorsal root ganglion neurons was detected using western blot assay. RESULTS AND CONCLUSION: (1) Compared with the KN93 control group, axonal regeneration of dorsal root ganglion neurons was inhibited, and the p-Smad1 protein expression was decreased in the KN93 experimental group, showing significant differences. (2) Compared with the CdCl2 control group, axonal regeneration of dorsal root ganglion neurons was promoted, and p-Smad1 protein expression was increased in the CdCl2 experimental group, showing significant differences. (3) The results showed that the CaMKII-Smad1 signaling pathway had a regulatory effect on axonal regeneration of dorsal root ganglion neurons.
ABSTRACT
Objective@#To investigate the role of the bone morphogenetic protein 2 (BMP2)⁃Smad1/5 and p38MAPK signaling pathways in the osteogenic differentiation of MSMSCs by insulin⁃like growth factor 1 (IGF1).@* Methods @#A re⁃ combinant adenovirus (RAD) and IGF1 expressing IGF1 gene were constructed. After osteogenic induction, qRT⁃PCR and Western blot were used to detect the phosphorylation level of Smad1/5 and the expression of the BMP⁃2 protein in the BMP⁃Smad signaling pathway; immunohistochemistry was used to observe the nuclear translocation of Smad1/5; qRT⁃PCR and Western blot were used to detect IGF with Noggin and SB203580, inhibitors of the p38MAPK signaling path⁃ way 1⁃mediated osteogenic differentiation of MSMSCs@* Results@#The recombinant IGF1 adenovirus was constructed suc⁃ cessfully. MSMSCs were cultured in inductive medium after infection with different concentrations of Ad⁃IGF1, and then, the protein levels of BMP2 and p⁃Smad1/5 increased. IGF1 can also induce nuclear translocation of Smad1/5. In addition, Noggin significantly reduced the phosphorylation level of Smad1/5 and the formation of mineralized nodules in the MSMSCs. The mRNA levels of Runx2, OPN and ALP also decreased. In contrast, SB203580 decreased neither the phosphorylation level of p38 nor the mRNA expression of Runx2, OPN and ALP in the Ad⁃IGF1 MSMSCs@* Conclu⁃sion@#IGF1 can promote the osteogenic differentiation of MSMSCs via the BMP2⁃Smad1/5 signaling pathway. In con⁃ trast, IGF1 may not promote the osteogenic differentiation of MSMSCs via the p38MAPK signaling pathway.
ABSTRACT
Peptides from Pilose antler aqueous extract (PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE's effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction (Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200 μg·mL showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.
ABSTRACT
PURPOSE: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. METHODS: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn− (1 µM Zn) or Zn+ (15 µM Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. RESULTS: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn−. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn−. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn−, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn−, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn−. CONCLUSION: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.
Subject(s)
Alkaline Phosphatase , Cell Line , Collagen Type I , Gene Expression , Osteoblasts , Osteocalcin , Osteopontin , Phosphorylation , Transcription Factors , ZincABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p.</p><p><b>METHODS</b>Cardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting.</p><p><b>RESULTS</b>Over-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts.</p><p><b>CONCLUSIONS</b>As the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.</p>
ABSTRACT
Objective:To clarify the effects of the BMP receptor inhibitor LDN-193189 in the dedifferentiated chondrosarcoma (DDCS) cell line NDCS-1 and to explore the anti-tumor mechanism of LDN-193189 in DDCS. Methods:NDCS-1 was treated with 5 nmol/L of LDN-193189. MTT assay and clone formation experiments were used to verify that LDN-193189 suppressed cel proliferation. Transwel and wound healing tests were performed to demonstrate that LDN-193189 inhibited cell invasion. Western blot detection was used to show that LDN-193189 inhibited the suppression of BMPR2, p-Smad1/5, and RUNX2 protein expression. Results:The BMPR2 signaling pathway was inhibited by LDN-193189;thus, cell viability and invasion were significantly suppressed. Conclusion:LDN-193189 induces the inhibition of progression in vitro via the BMPR2-p-Smad1/5-RUNX2 signaling pathway in the human DDCS cell line NDCS-1.
ABSTRACT
Diabetic nephropapthy (DN) is a common cause of end-stage renal disease.Early diagnosis and treatment are significant for delaying the development of DN.Smad proteins mediate transforming growth factor β superfamily intracellular signal transduction.Recent studies have found that Smad1 protein plays an important role in the process of renal fibrosis.Urine Smad1 emerges in the early stage of DN,which is expected to be a non-invasive indicator in early diagnosis of DN.
ABSTRACT
BACKGROUND: Follicular lymphomas present with various immunohistologic patterns. The immunohistochemical markers used in the diagnosis of follicular lymphoma show variable degrees of sensitivity and specificity, and thus, additional germinal center markers are required. Smad1 has been reported to be overexpressed in follicular lymphoma, but little is known regarding the expression patterns of Smad proteins in human lymphoid tissue. METHODS: In the present study, we performed immunohistochemistry for traditional germinal center markers and for Smad1 in human reactive lymphoid and follicular lymphoma tissues to investigate Smad1's usefulness in the diagnosis of follicular lymphoma. RESULTS: In the reactive germinal centers, most cells were positive for Smad1. Among the 27 follicular lymphoma cases, 17 of 21 (80%) were Smad1 positive, 17 of 27 (63%) were positive for CD10, and 23 of 27 (85%) were positive for Bcl6. Notably, three cases expressed CD10 only, and one only expressed Bcl6. All these cases were grade 3 tumors and showed follicular and diffuse growth patterns. CONCLUSIONS: These results indicate that Smad1 is a candidate as a germinal center marker. Furthermore, they suggest that the Smad signaling pathway might be involved in follicular lymphoma.
Subject(s)
Humans , Diagnosis , Germinal Center , Immunohistochemistry , Lymphoid Tissue , Lymphoma , Lymphoma, Follicular , Sensitivity and Specificity , Smad ProteinsABSTRACT
This study was aimed to observe four kinds of kidney-tonification medicine, which were Epimedium, pso-ralen, Ligustrum lucidum, Polygonum with the active ingredient of icariin, psoralen, oleanolic acid, stilbene glucoside and their orthogonal compatibility. There were two kinds of non-kidney tonification medicine, which were Chuanx-iong and astragalus with the active ingredient of TMP and astragaloside. The observation was made on the regulatory role of rat bone marrow stem cells (BMSCs). A total of 65 SD rats were randomly divided into the normal control group, positive transformed control group, kidney-tonification compatibility group (including Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, Group 7, Group 8, and Group 9), non-kidney tonification medicine control group (in-cluding TMP group and astragaloside group). Intragastric administration of medication was given to the kidney-tonifi-cation compatibility group and the non-kidney tonification medicine control group, once a day for 3 consecutive days. Intragastric administration of equal amount of normal saline was given to the normal control group and the posi-tive transformed control group. On the third day of intragastric administration, rats in each group were sacrificed. Serum containing medication was used in the culture of BMSCs for 6, 12, or 18 days. ELISA method was used to quantitatively detect the expression activity and content of BMP2 on the 6th, 12th, or 18th day, in order to evaluate the degree of bone cell differentiation degree. Real-time quantitative PCR method was used for detection of expression of Bmp2, Smad1, Smad4 mRNA in serum containing medication in the culture of BMSCs on the 18th day. The results showed that the kidney-tonification compatibility can improve the expression activity and content of BMP2 culture in vitro, with the peak on the 12th day. The kidney-tonification compatibility groups can upregulate expressions of Bmp2, Smad1, Smad4 mRNA. It was concluded that the active ingredient compatibility of kidney-tonification medicine can promote BMSCs. Its mechanism may be related to the upregulation of expression of Bmp2, Smad1, Smad4 mRNA, and the activity and content of Bmp2.
ABSTRACT
Objective:To clone and sequence cDNA of MH2 domain of Smad1 gene from human dental papilla cells. Methods:Total RNA was isolated from primarily cultured human dental papilla cells and reversely transcribed into single stranded cDNA.The desired DNA product was obtained by PCR with two gene specific primers.The segment was inserted into PUC19 vector and the plasmid was transformed into E.coli JM109.The double stranded cDNA of positive clone was sequenced.Results:The sequence of MHz domain of Smad1 cloned from human dental papilla cells was consistent with that reported by Hoodless et al. Conclusion:Smad1 exists in human dental papilla cells,BMP signaling may be mediated by smad1 in human dental papilla cells.