ObjectiveTo summarize the research progress on the calcitonin gene-related peptide (CGRP) and receptor activator of nuclear factor κB (RANK)/receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) system during bone reconstruction to provide theoretical basis for further research on the prevention and treatment of bone-related diseases.MethodsThe relevant research results at home and abroad in recent years were analyzed and summarized.ResultsCGRP and RANK/RANKL/OPG system play important regulatory roles in the bone reconstruction.ConclusionAt present, the research on the mechanism of CGRP and RANK/RANKL/OPG system in bone reconstruction is insufficient. Therefore, it is necessary to study further on the process and interrelation of CGRP and RANK/RANKL/OPG system in bone reconstruction to confirm their mechanism, which will bring new ideas and methods for the treatment of bone related diseases in clinic.
ObjectiveTo explore the effect and mechanism of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice.MethodsThe microglia were isolated and purified from brain tissue of new-born BALB/c mice through differential velocity adherent and vibration technique. The quantity of the microglia was identified by immunofluorescence staining of inducible nitric oxide synthetase (iNOS) and ionized calcium binding adapter molecule 1 (Iba1) and real-time fluorescence quantitative PCR (qRT-PCR) for special expression genes [iNOS, CD32, and interlenkin 10 (IL-10)]. Then the microglia were cultured with SN50, and the expressions of nuclear factor κB (NF-κB), differentiation-related genes (iNOS, CD11b, IL-10, and CD206), and apoptosis were detected by Western blot, qRT-PCR, and flow cytometry, respectively. The hypoxia model of neuron was established, and the cell apoptosis was evaluated by MTT after 0, 2, 6, 12, 24, and 48 hours of anoxic treatment. The apoptosis related markers (Bcl-2 and Caspase-3) were measured by Western blot and flow cytometry. In addition, the neurons after anoxic treatment were co-cultured with SN50 treated microglia (experimental group) and normal microglia (control group) for 24 hours. And the cell viability and apoptosis related markers (Bcl-2 and Caspase-3) were also measured.ResultsImmunofluorescence staining and qRT-PCR analysis showed that the cells expressed the specific proteins and genes of microglia. Compared with the normal microglia, the relative expressions of NF-κB protein and iNOS and CD11b mRNAs in the microglia treated with SN50 significantly decreased (P<0.05), the relative expressions of IL-10 and CD206 mRNAs significantly increased (P<0.05), and the cell apoptosis rate had no significant change (P>0.05). Compared with the normal neurons, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins after anoxic treatment significantly decreased (P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate of neurons significantly increased (P<0.05). In the co-culture system, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins were significantly higher in experimental group than those in control group (P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate were significantly lower in experimental group than those in control group (P<0.05).ConclusionSN50 can induce the microglia differentiation into M2 type through NF-κB pathway. The SN50-induced microglia can protect neurons from hypoxic injury.
ObjectiveTo explore the effect and potential mechanism of glycyrrhizin (GL) by inhibiting high mobility group box 1 (HMGB1) on glial scar formation after spinal cord injury (SCI) in rats.MethodsSeventy-two female Sprague Dawley rats were randomly divided into sham group (n=12), SCI model group (SCI group, n=36), GL intervention group (SCI+GL group, n=12), and nuclear factor κB (NF-κB) inhibitor [pynolidine dithiocarbamate (PDTC)] intervention group (SCI+PDTC group, n=12). The SCI models of SCI group, SCI+GL group, and SCI+PDTC group were made by modified Allen’s method, the sham group was only exposed the spinal cord without any injury. First of all, Basso-Beattie-Bresnahan (BBB) score of hind limbs and slope test were performed in SCI group at 1, 2, and 3 weeks after operation; Western blot was used to detect the expressions of glial fibrillary acidic protein (GFAP) and HMGB1 proteins. Compared with the sham group, the most significant time point in the SCI group was selected for subsequent experiment, in which the most significant glial scar was formed. Then, behavioral tests (BBB score of hind limbs and slope test), histological observation of spinal cord tissue structure, Western blot detection of HMGB1, GFAP, and NF-κB proteins, and immunohistochemical staining observation of GFAP and chondroitin sulfate proteoglycan (CSPG) were used to explore the effect of GL on the formation of glial scar after SCI and its potential mechanism.ResultsThe BBB score and slope angle of the SCI group increased gradually with time, which were significantly lower than those of the sham group at each time point (P<0.05). Western blot detection showed that the relative expressions of HMGB1 and GFAP proteins in the SCI group at 1, 2, and 3 weeks after operation were significantly higher than those in sham group (P<0.05). The change was most obvious at 3 weeks after SCI, therefore the spinal cord tissue was selected for subsequent experiments at this time point. At 3 weeks after operation, compared with the SCI group, BBB score and slope angle of SCI+GL group significantly increased (P<0.05); the relative expressions of HMGB1, GFAP, and NF-κB proteins detected by Western blot and the expressions of GFAP and CSPG proteins detected by immunohistochemical staining significantly decreased (P<0.05); the disorder of spinal cord tissue by HE staining improved, inflammatory cell infiltration reduced, and glial scar formation decreased. At 3 weeks after operation, the expressions of NF-κB, GFAP, and CSPG proteins of the SCI+PDTC group significantly reduced when compared with the SCI group (P<0.05); and the expression of NF-κB protein significantly decreased and the expressions of GFAP and CSPG proteins significantly increased when compared with the SCI+GL group (P<0.05).ConclusionAfter SCI in rats, the application of GL to inhibit the expression of HMGB1 can reduce the expression of GFAP and CSPG in the injured spinal cord, then reduce the formation of glial scars and promote the recovery of motor function of the hind limbs, and GL may play a role in inhibiting glial scar through HMGB1/NF-κB pathway.
ObjectiveTo investigate the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits.MethodsTwenty-six 3-month-old New Zealand rabbits, 2.0-2.5 kg in weight, were used to establish the model of intervertebral disc degeneration at L3, 4, L4, 5, and L5, 6 by transabdominal needle puncture. At 4 weeks after operation, the 24 rabbits were randomly divided into 4 groups after successful modeling, which checked by MRI. The target intervertebral discs of each group were injected with zinc finger protein A20 overexpressed adenovirus (Ov-A20 group), empty carrier adenovirus (NC group), phosphate buffer saline (control group), and shRNA-A20 adenovirus (Sh-A20 group). The biological responses of animals in each group were comprehensive scored before 1 day of injection and after 1, 2, 3, and 6 days of injection. At 2, 4, and 8 weeks after injection, the animals in each group were observed by MRI to obtain the exact T2 relaxation time (T2 signal value). After MRI examination, the animals were killed to take the degenerative intervertebral disc tissue; and the tissue was detected by Alcian blue staining to observed the intervertebral disc degeneration. The expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were detected by immunohistochemistry staining. The expressions of zinc finger protein A20, nuclear factor κB binding protein [P65, phosphate P65 (P-P65), collagen Ⅱ, aggrecan], inflammatory factors [tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β)], autophagy-related protein [LC3 (LC3Ⅱ/LC3Ⅰ) and P62] were detected by Western blot.ResultsThe comprehensive score of biological response in each group after injection was significantly lower than that before injection (P<0.05). At 6 days after injection, the comprehensive score of biological response in the Sh-A20 group was significantly lower than that in other groups (P<0.05), and there was no significant difference among other groups (P>0.05). The detection of MRI showed that the T2 signal value in the Ov-A20 group was the highest at 2, 4, and 8 weeks after injection (P<0.05), and the T2 signal value in the Sh-A20 group was the lowest at 2 and 4 weeks after injection (P<0.05). There was no significant difference between other groups (P>0.05). Alcian blue staining showed that the expression of aggrecan was the highest in Ov-A20 group and the lowest in Sh-A20 group at 4 weeks (P<0.05); the expression of aggrecan in Ov-A20 group was the highest at 8 weeks (P<0.05), and there was no significant difference between other groups (P>0.05). Immunohistochemical staining showed that the expressions of zinc finger protein A20, collagen Ⅱ, and aggrecan were the highest in Ov-A20 group and lowest in Sh-A20 group (P<0.05). Western blot showed that the expressions of zinc finger protein A20, collagen Ⅱ, aggrecan, and LC3 (LC3Ⅱ/LC3Ⅰ) proteins were the highest in the Ov-A20 group and the lowest in Sh-A20 group (P<0.05), while the expressions of P-P65, TNF-α, IL-1β, and P62 proteins were the lowest in Ov-A20 group and the highest in Sh-A20 group (P<0.05). There was no significant difference in the expression of p65 protein between groups (P>0.05).ConclusionZinc finger protein A20 can effectively regulate the process of lumbar intervertebral disc degeneration in rabbits by inhibiting inflammation.