| Objective1.To explore the molecular mechanism of long non-coding RNA-DANCR regulating nuclear factor-κB(NF-κB)bone immune pathway in Postmenopausal osteoporosis(PMOP).2.To explore whether quercetin regulates the NF-κB bone immune pathway by mediating DANCR,thus delaying bone loss of PMOP.Methods1.To explore the molecular mechanism of DANCR regulating NF-κB bone immune pathway in PMOP from clinical,animal and cellular levels.(1)The expression of DANCR in the serum of PMOP patients,bone marrow of ovariectomized mice and the osteoclast differentiation of mouse mononuclear macrophage cell line RAW264.7.① Serum samples of PMOP and normal bone mass patients were collected from 20 patients in each group.The expression of DANCR in serum of the two groups was detected by RT-qPCR.② Twelve 8-week-old C57 mice were randomly divided into two groups:sham operation group and model group,with 6 mice in each group.In the model group,the PMOP model was constructed by bilateral ovariectomy.In the sham group,only the adipose tissue of the same size as the ovary was removed.After 12 weeks,bone marrow from bilateral femurs of mice in both groups was collected,and the expression of DANCR in bone marrow of mice in both groups was detected by RT-qPCR.③ The mouse monocyte macrophage line RAW264.7 was induced to differentiate into osteoclasts using 10ng/ml RANKL,and DANCR expression at 0,2 and 4 days were detected.(2)Effect of DANCR knockout on osteoclast differentiation of RAW264.7 cells① The CRISPR/Cas9 technique was used to knock out DANCR gene in RAW264.7 cells,and finally two cell lines with DANCR gene knockout effect G1 and F5 were produced.②The effect of DANCR knockout on RAW264.7 cells was verified by RT-qPCR.③ The proliferation ability of RAW264.7 cells in WT,Gl and F5 groups was detected by CCK8 method at 0,24,48 and 72 hours.④ The osteoclast differentiation ability of RAW264.7 cells in WT,G1 and F5 groups was detected by TRAP staining.⑤ The expressions of osteoclast-related marker proteins NFATC1,DC-STAMP and CTSK in WT,G1 and F5 after osteoclast induction were detected by Western blot.(3)The effect of DANCR knockout on the NF-κB bone immune pathway and the reversing effect of Lipopolysaccharide(LPS),an activator of NF-κB bone immune pathway① The expression of p-P50/P50,the key protein of NF-κB bone immune pathway,was detected by Western blot in WT,G1 and F5 groups before and after RANKL induction.②WT,G1 and F5 cells induced by RANKL were treated with 500ng/ml LPS.TRAP staining was used to detect the osteoclast differentiation ability of cells in each group,and Western blot was used to detect the protein expressions of NFATC1,DC-STAMP,CTSK,p-P50 and P50 of cells in each group.(4)Effect of DANCR on bone mass and NF-κB bone immune pathway in PMOP mouse model① The C57 female mice with systemic DANCR knockout were constructed by CRISPR/Cas9 technique,and 7 DANCR knockout homozygous mice(DANCR-/-)and 7 litternegative mice(CON)were obtained.② Both CON and DANCR-/-groups underwent bilateral ovariectomy to establish PMOP model at 8 weeks,and sampling was performed at 12 weeks after surgery.③ Micro-CT was used to detect the micro-structure of lumbar vertebrae.The region of interest of lumbar cancellous bone was selected and the 3D reconstruction map was constructed.The parameters of bone micro-structure were obtained,including the bone volume/total volume(BV/TV,%),trabecular number(Tb.N,1/mm),trabecular thickness(Tb.Th,mm),and trabecular separation(Tb.Sp,mm).④ The histological morphology of lumbar vertebrae was detected by HE staining.⑤ TRAP staining was used to detect osteoclast generation in lumbar vertebrae,and the ratio of osteoclast number/bone surface(Oc.N/BS)and ratio of osteoclast surface/bone surface(Oc.S/BS)were calculated.⑥ The expression of p-P50/P50 in lumbar vertebrae was detected by Western blot.2.To explore whether the effective mechanism of quercetin regulating the NF-κB bone immune pathway by mediating DANCR at the animal and cellular levels,thus delaying bone loss of PMOP(1)Therapeutic effect of quercetin on PMOP①Twenty-one 8-week-old female C57 mice were randomly divided into Sham group(Sham),model group(OVX)and quercetin group(OVX+Q),with 7 mice in each group.OVX and OVX+Q groups underwent bilateral ovaries resection to prepare PMOP model,and sham operation group underwent sham operation.12 weeks after surgery,OVX+Q group was given 50mg/kg quercetin gavage(5%sodium carboxymethyl cellulose was the solvent),Sham and OVX groups were given the same amount of 5%sodium carboxymethyl cellulose gavage.All groups were given the drug once a day for 6 weeks.② Micro-CT was used to detect the micro-structure of lumbar vertebrae.After selecting the region of interest of lumbar cancellous bone,the bone micro-structure parameters BV/TV,Tb.N,Tb.Th and Tb.Sp were obtained.③ The tissue morphology of lumbar vertebrae was observed by HE staining.④ TRAP staining was used to observe osteoclast formation in lumbar vertebrae,and Oc.N/BS and OC.S/BS were calculated.⑤ The expression of DANCR in lumbar vertebrae was detected by RT-qPCR.⑥ The expression of p-P50/P50 in lumbar vertebrae was detected by Western blot.(2)The effect of quercetin on osteoclast formation① Bone marrow-derived macrophages(BMMs)were extracted from mouse femur and tibia,and macrophage colony stimulating factor(M-CSF)and receptor activator of nuclear factor κB ligand(RANKL)were combined to induce differentiation into mature osteoclasts.The effects of quercetin at different concentrations(0μM,0.1 μM,0.5μM,1μM,5μM,10μM)on BMMs proliferation at 24,48,72,96 h were determined by CCK8 method,and the intervention concentrations that did not affect BMMs proliferation were screened.② TRAP staining was used to detect the effect of quercetin on osteoclast differentiation of BMMs.③ Hydroxyapatite coating was used to detect the effect of quercetin on bone resorption after osteoclastic differentiation of BMMs.④ The influence of quercetin on the expression of NFATC1,DC-STAMP,CTSK,pP50 and P50 proteins during osteoclast differentiation of BMMs was detected by Western blot.⑤The effect of quercetin on DANCR expression in osteoclast differentiation of BMMs was detected by RT-qPCR.Results:1.The molecular mechanism of DANCR regulating NF-κB bone immune pathway in PMOP(1)DANCR was highly expressed in the serum of PMOP patients,bone marrow of ovariectomized mice and the osteoclast differentiation of mouse mononuclear macrophage cell line RAW264.7① There were 20 patients in serum in the normal bone mass group and 20 patients in the PMOP group,and there were no statistical differences in age,menopause age and menopause years between the two groups(P>0.05,P>0.05,P>0.05).Bone mineral density and T value in the PMOP group were significantly decreased compared with the normal bone mass group(P<0.01,P<0.01).Compared with patients with normal bone mass,serum DANCR expression in the PMOP group was significantly upregulated(P<0.01).② Compared with Sham group,DANCR expression in bone marrow of OVX group was significantly up-regulated(P<0.01).③ DANCR expression during osteoclast differentiation of RAW264.7 was detected.Compared with day 0,DANCR expression was significantly higher on day 2(P<0.01).Compared with day 2,DANCR expression was significantly increased on day 4(P<0.05).(2)DANCR knockout inhibited osteoclast differentiation of RAW264.7 cells① DANCR knockout effect of RAW264.7 cells was successful.Compared with WT group,DANCR expression in G1 and F5 group was significantly decreased(P<0.01,P<0.01).② There was no significant difference in proliferation ability of RAW264.7 cells in WT,G1 and F5 groups at 0,24,48 and 72 hours,indicating that DANCR knockout does not affect the proliferation of RAW264.7.③ DANCR knockout inhibited osteoclast differentiation of RAW264.7 cells.Compared with WT group,the number and area of osteoclasts in G1 group were significantly decreased(P<0.01,P<0.01),and the area of osteoclasts in F5 group was significantly decreased(P<0.01).④In WT group,compared with non-RANKL-induced group,the protein expressions of CTSK,NFATC1 and DC-STAMP were significantly increased after RANKL induction(P<0.01,P<0.01,P<0.01).In G1 group,compared with the non-RANKLinduced group,there was no statistical difference in the protein expression of CTSK and DC-STAMP after RANKL induction,while the protein expression of NFATC1 decreased significantly(P<0.01).In F5 group,compared with non-RanKL-induced group,CTSK and NFATC1 protein expressions were significantly decreased after RANKL induction(P<0.01,P<0.01),while DC-STAMP showed a downward trend.The protein expression in WT,G1 and F5 groups were compared under RANKL induction.Compared with WT group,the protein expressions of CTSK,NFATC1 and DC-STAMP in G1 group were significantly decreased(P<0.01,P<0.01,P<0.01),and the protein expressions of CTSK,NFATC1 and DC-STAMP in F5 group were also significantly decreased(P<0.01,P<0.01,P<0.01).(3)DANCR knockout inhibited the activation of NF-κB bone immune pathway in osteoclast differentiation of RAW264.7,while LPS could reverse the process① In WT group,the ratio of P-P50/P50 after RANKL induction was significantly higher than that in non-RanKL-induced group(P<0.01).Compared with the nonRanK1-induced group,the P-P50/P50 ratio was significantly decreased in G1 group after RANKL induction(P<0.01,P<0.01),while there was a downward trend in F5 group after RANKL induction.The comparison between groups of WT,G1 and F5 cells after RANKL induction showed that compared with WT group,the P-P50/P50 ratio in G1 group was significantly decreased(p<0.01),and F5 group showed a downward trend.② In WT group,TRAP staining was significantly deeper and the number and area of osteoclasts were significantly increased after addition of LPS compared with non-LPS group(P<0.01,P<0.01).In Gl or F5 groups,TRAP staining was also significantly deeper and the number and area of osteoclasts were significantly increased after addition of LPS compared with non-LPS groups(P<0.01,P<0.01).③ In WT,G1 or F5 groups,the ratio of p-P50/P50 protein was significantly increased after adding LPS compared with non-LPS group,with statistical differences(P<0.05,P<0.05,P<0.05).④ In WT,G1 or F5 groups,compared with non-LPS group,osteoclast marker proteins CTSK,NFATC1 and DC-STAMP were significantly increased after the addition of LPS,with statistical differences(P<0.05,P<0.05,P<0.05).(4)DANCR knockout inhibited bone loss and activation of NF-κB bone immune pathway in PMOP mouse model① The three-dimensional reconstruction of lumbar cancellous bone showed that compared with the CON group,the bone mass of lumbar cancellous bone in DANCR/-group was significantly increased,and B V/TV and Tb.N were significantly increased(P<0.05),while Tb.Sp had a downward trend.② HE staining showed that the bone trabeculae in the CON group were sparse,fractured and thin,while the number of bone trabeculae in the DANCR-/-group was more and thicker than that in the CON group,and the fracture was significantly reduced.③ Compared with CON group,Oc.N/BS and Oc.S/BS in lumbar vertebrae of DANCR/-group were significantly decreased(P<0.01,P<0.01).④Compared with CON group,the protein expression ratio of p-P50/P50 in DANCR/-group was significantly decreased(P<0.01).2.Quercetin down-regulated DANCR to inhibit NF-κB bone immune pathway and delayed bone loss of PMOP(1)Therapeutic effect of quercetin on PMOP① Three-dimensional reconstruction of lumbar cancellous bone in micro-CT showed that bone mass was significantly lost in OVX group compared with Sham group,while bone mass was significantly increased in OVX+Q group compared with OVX group.Compared with Sham group,BV/TV and Tb.N were significantly decreased(P<0.01,P<0.01),Tb.Sp was significantly increased(P<0.01),and Tb.Th had no significant change(P>0.05).Compared with OVX group,BV/TV in OVX+Q group was significantly increased(P<0.05),Tb.N was significantly increased(P<0.01),Tb.Sp was significantly decreased(P<0.01),and Tb.Th had no significant difference(P>0.05).② HE staining showed that compared with Sham group,OVX group had more trabecular distance,thinner trabecular,more fracture and poor continuity.Compared with OVX group,OVX+Q group had more trabeculae and less fracture.③ TRAP staining showed that,compared with Sham group,deeper TRAP staining was observed in OVX group,and Oc.N/BS and Oc.S/BS were significantly increased(P<0.01,P<0.01),indicating increased osteoclast generation.Compared with OVX group,OVX+Q group showed shallower TRAP staining,and Oc.N/BS and Oc.S/BS were significantly decreased(P<0.01,P<0.01),indicating less osteoclast generation.④ Compared with Sham group,DANCR in bone tissue of OVX group was significantly up-regulated(P<0.01).Compared with OVX group,DANCR in bone tissue of OVX+Q group was significantly down-regulated(P<0.01).⑤ Compared with Sham group,the ratio of p-P50/P50 in bone tissue of OVX group was significantly increased(P<0.01).Compared with OVX group,the ratio of p-P50/P50 in bone tissue of OVX+Q group was significantly decreased(P<0.01).(2)The effect of quercetin on osteoclast formation① When the quercetin concentration was less than 0.5μM,the BMMs proliferation was neither promoted nor inhibited.②With the increase of quercetin concentration(0,0.125uM,0.25uM,0.5uM),the number and area of BMMs differentiation into osteoclasts were significantly decreased(P<0.01,P<0.01),and the area of bone lacunae formation was significantly decreased(P<0.01).③ Quercetin at different concentrations could significantly inhibit the expression of osteoclast-related protein CTSK,DC-STAMP and NFATC1(P<0.05,P<0.01,P<0.01),and the trend of inhibiting osteoclast-related protein expression was more obvious with the increase of quercetin concentration.④Compared with Con group,DANCR expression in 0.125uM,0.25uM and 0.5uM quercetin groups was significantly decreased(P<0.01,P<0.01,P<0.01),and showed a trend of gradual decline with the increase of concentration.⑤Quercetin significantly decreased p-P50/P50 protein ratio in osteoclast differentiation of BMMs at 15min and 30min(P<0.05,P<0.05).ConclusionThis study confirmed that DANCR regulates the NF-κB bone immune pathway at the clinical,animal and cellular levels as an important molecular mechanism of PMOP pathogenesis,and DANCR can be used as a novel target for PMOP prediction.Based on this mechanism,it is confirmed that quercetin can inhibit the bone resorption caused by osteoclasts by down-regulating DANCR and NF-κB bone immune pathway,and finally play a role in delaying the bone loss of PMOP. |
PDF Full Text Request