Experimental Study On The Effect Of Theaflavin-3,3'-bisgallate On The Regulation Of Macrophage Polarization On Inflammatory Bone Loss

Part I:Effect of theaflavin-3,3'-digallate(TF3)on macrophages in TNF-?-induced inflammatory conditionsObjective:To investigate the role of TF3 in promoting the transformation of macrophage to M2 type under inflammatory conditionsMethods:The effects of TF3 on TNF-?-induced inflammatory conditions macrophage polarization were observed in bone marrow-derived macrophages(BMMs)of mice.Cell Counting Kit-8 test(CCK8 test)was used for cell proliferation and toxicity analysis.BMMs were divided into control group,TNF-? group,TNF-?+low-TF3 group and TNF-?+high-TF3 group.The phenotypic expression of macrophages was detected by cell polarization staining.The expression of inflammatory factors was detected by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative reverse transcriptase polymerase chain reaction(Real-time quantitative PCR)was used to detect proinflammatory cytokine gene IL-1?,IL-6,M1 type macrophage marker iNOS,and anti-inflammatory cytokine gene IL-10 and M2 macrophage marker arginse-1 were detected.Results:The results of CCK8 test showed that when the concentration of TF3 was 0.01-10 ?M,it had no significant effect on the proliferation of BMMs cells and had no toxic effect on the cells.The results of immunofluorescence double staining of F4/80 and iNOS,and F4/80 and Arg-1 cells showed that the expression level of iNOS in macrophages stimulated by TNF-? was significantly higher than that in the control group,while the expression level of Arg-1 in macrophages was significantly lower than that in the control group.This showed that TNF-? can induce macrophage polarization to M1 type and inhibit polarization to M2 type.However,after TNF-? stimulation and TF3 intervention,the expression level of iNOS decreased and the expression of Arg-1 increased,suggesting that TF3 can inhibit macrophage polarization to M1 type and promote macrophage polarization to M2 type in a dose-dependent manner.The contents of IL-1?,IL-6,NO and IL-10 in the supernatant of cell culture in vitro were detected by ELISA test.The results showed that the concentrations of IL-1?,IL-6 and NO in macrophages stimulated by TNF-a were significantly higher than those in the control group,but the concentration of IL-10 decreased.After the intervention of TF3,the concentrations of IL-1?,IL-6 and NO in the supernatant decreased,while the concentration of IL-10 increased in a dose-dependent manner.These results suggest that TF3 can effectively inhibit the secretion of proinflammatory cytokines and promote the secretion of anti-inflammatory cytokines,so as to inhibit the pro-inflammatory response of macrophages and promote the anti-inflammatory response of macrophages.The expression of pro-inflammatory cytokine genes IL-1?,IL-6 and iNOS and anti-inflammatory cytokine genes Arg-1 and IL-10 were detected by RT-PCR.The results showed that compared with Control group,the expression levels of pro-inflammatory cytokine genes IL-1?,IL-6 and iNOS were significantly increased,while the expression levels of anti-inflammatory cytokine genes Arg-1 and IL-10 were decreased.However,after the intervention of TF3,the expression level of proinflammatory cytokine gene was significantly decreased and the expression level of anti-inflammatory cytokine was increased.Moreover,the inhibitory effect of TF3 on proinflammatory cytokine gene expression and the promotion of anti-inflammatory cytokine gene expression were positively correlated with the intervention concentration of TF3.Conclusion:TF3 can inhibit the polarization of macrophages to M1 type and promote the polarization of macrophages to M2 type.TF3 can inhibit the secretion of proinflammatory cytokines and promote the secretion of anti-inflammatory cytokines,thus playing an anti-inflammatory role.TF3 can inhibit the expression of proinflammatory cytokine gene and promote the expression of anti-inflammatory cytokine gene.The above effects of TF3 were dose-dependent.Part ?:The effect of TF3 on the inhibition of osteogenic differentiation of MC3T3-E1 cells induced by TNF-?Objective:To investigate the effect of TF3 on osteogenic differentiation of MC3T3-E1 cells under inflammatory conditions.Methods:Mouse embryonic osteoblast progenitor cells(MC3T3-E1)were used to observe the differentiation and maturation of osteoblast progenitor cells induced by chronic inflammation in vitro.CCK8 test was used to analyze cell proliferation and toxicity.The cells were divided into four groups:control group,TNF-? group,TNF-?+low-TF3 group and TNF-?+high-TF3 group.The activity of osteoblasts was detected by alkaline phosphatase(ALP)staining,the mineralization of osteoblasts was observed by alizarin red staining,and the osteocalcin(OCN)was detected by phalloidin fluorescence staining.The mRNA expression levels of osteoblast related genes RUNX2,ALP and OCN were detected by RT-PCR.Results:The results of CCK8 test showed that when the concentration of TF3 was 0.01-10 ?M,it had no effect on the proliferation of MC3T3-E1 cells.The results of ALP staining showed that TF3 could alleviate the inhibitory effect of TNF-? on alkaline phosphatase activity of MC3T3-E1 cells to a certain extent.The results of alizarin red staining showed that TNF-? could significantly inhibit the formation of calcium minerals during osteogenesis,and in the TF3 intervention group in the concentration range of 0.01?M,the formation of calcium minerals increased with the increase of TF3 concentration.The results of immunofluorescence staining of phalloidin showed that TNF-? could inhibit the expression of osteogenic associated proteins,but in the concentration range of 0.01-1?M,TF3 could reduce the inhibitory effect of TNF-? to a certain extent.RT-PCR results showed that TNF-? could significantly inhibit the mRNA expression of osteogenic related genes RUNX2,ALP and OCN.Compared with the TNF-? group,in the concentration range of 0.1?M-1 ?M,the expression of these mRNA increased in a concentration-dependent manner with the increase of TF3 concentration.Conclusion:In the culture of osteoblasts under the condition of inflammation in vitro,TF3 can alleviate the inhibitory effect of inflammatory factors on osteogenic activity and improve the effect of inflammatory factors on osteogenic activation in a concentration-dependent manner.TF3 could also reduce the inhibitory effect of inflammatory factors on the expression of osteogenesis-related proteins and osteogenesis-related genes.Part ?:The therapeutic effect of TF3 on bone loss in rheumatoid arthritisObjective:To investigate the therapeutic effect of TF3 on bone mass loss in Collagen-Induced Arthritis(CIA)mice.Methods:32 male 8-week-old DBA/1 mice were randomly divided into 4 groups(n=8 in each group):blank control group(Sham group).model experimental group(Vehicle group),low dose TF3 treatment group(Low-TF3 group),high dose TF3 treatment group(High-TF3 group).Except for the Sham group without any treatment,the other three groups were immunized with bovine type ? collagen one day before the second collagen challenge(Day 20).Low-TF3 group and High-TF3 group were injected intraperitoneally with low dose 1mg/kg/day and high dose 10mg/kg/day TF3 respectively for 6 days/week for 8 weeks.Sham group and Vehicle group were injected intraperitoneally with the same amount of normal saline respectively.After administration,the body weight,arthritis score and paw thickness of mice were recorded twice a week.At the end of the fifth week,the mice were killed and eyeball blood the liver and kidney tissue,bilateral knee joints and feet were taken.The three-dimensional reconstruction of feet was analyzed by micro-computerized tomography(Micro-CT),the degree of inflammatory cell infiltration was evaluated by H&E staining,and the destruction of knee cartilage was evaluated by Safranin O-Fast green cartilage staining.The expressions of TNF-?,IL-10,OCN and Runx2 were detected by immunohistochemical staining,and the polarization of macrophages was evaluated by immunofluorescence staining.The peripheral blood levels of TNF-?,IL-1?,IL-6 and IL-10 in were measured by ELISA,and the liver and kidney tissue sections were stained with H&E to evaluate the injury of liver and kidney.Results:From D21,the body weight of mice in Sham group gradually increased,while the body weight of mice in Vehicle group,Low-TF3 group and High-TF3 group decreased,and the difference was statistically significant compared with Sham group.From D35,the body weight of mice in Low-TF3 group and High-TF3 group gradually increased,and the body weight of mice in Low-TF3 group and High-TF3 group was significantly higher than that in Vehicle group(p<0.05)at D42.The joint inflammation score was scored from D21 and the results showed that the joint inflammation score of Vehicle group reached the peak at D42.The arthritis score of Vehicle group was 13.8 ± 1.65 points.The mice in Low-TF3 group and High-TF3 group with no arthritis score of 16 points at D42,and the arthritis score decreased gradually decreased from D49,and the difference was statistically significant compared with Vehicle group(p<0.05).The results of Micro-CT showed that the joint structure of Sham group was intact,the destruction and deformity of joint structure in Vehicle group was the most serious,the joint deformity in Low-TF3 group was better than that in Vehicle group,and the joint space in High-TF3 group was slightly improved.The results of H&E staining showed that the number of bone trabeculae of knee joint in mice treated with TF3 was significantly higher than that in Vehicle group,the inflammatory reaction was alleviated,the inflammatory cells were significantly decreased,and the destruction of bone trabeculae was significantly improved.The degree of improvement was significantly dose-dependent.The results of Safranin O-Fast green cartilage staining showed that TF3 could significantly reduce the degree of articular cartilage damage in CIA mice,and could reduce the cartilage damage in CIA mice in a dose-dependent manner.The related markers of osteoblasts in CIA mice were detected by immunohistochemical staining.The results showed that the number of OCN and Runx2 positive cells in TF3 group was significantly higher than that in Vehicle group in a dose-dependent manner.The results of TNF-? and IL-10 detected by immunohistochemical staining showed that the expression of proinflammatory factor TNF-? decreased and the expression of anti-inflammatory factor IL-10 increased in CIA mice treated with TF3 and the degree was significantly dose-dependent.The results of immunofluorescence staining showed that TF3 reversed some macrophages from M1 type to M2 type and inhibited inflammation.The results of ELISA showed that TF3 could inhibit the expression of proinflammatory cytokines TNF-?,IL-1??IL-6 in CIA mice and promote the expression of anti-inflammatory cytokines IL-10 in CIA mice and the effect was dose-dependent.The results of H&E staining in the sections of liver and kidney of each group showed that there was no significant damage to the liver and kidney of mice treated with TF3 for 5 weeks.Conclusion:TF3 can effectively inhibit the expression of proinflammatory cytokines in CIA mice,promote the polarization of macrophages from M1 to M2 type,reduce inflammatory reaction and effectively reduce bone mass loss.It is expected to be an ideal drug for the prevention and treatment of rheumatoid arthritis.Part ?:The therapeutic effect of TF3 on osteoporotic bone lossObjective:To investigate the therapeutic effect of TF3 on bone mass loss in ovariectomized mice.Methods:A total of 32 female 8-week-old C57BL/J6 mice were randomly divided into 4 groups(n=8 in each group):sham operation group(Sham group),bilateral ovariectomy group(ovariectomy,OVX group).OVX with low dose TF3 treatment group(Low-TF3 group)and OVX with high dose TF3 treatment group(High-TF3 group).Sham group received only Sham surgery,no bilateral ovariectomy,no TF3,and received intraperitoneal injection of the same amount of normal saline.OVX group underwent bilateral ovariectomy without intraperitoneal injection of the same amount of normal saline,OVX group received bilateral ovariectomy with intraperitoneal injection of the same amount of normal saline.In Low-TF3 group,bilateral ovariectomy was performed.After operation,low dose 1mg/kg/day TF3 was injected intraperitoneally for 6 days/week for 8 weeks.In High-TF3 group,bilateral ovariectomy was performed.After operation,low dose 10mg/kg/day TF3 was injected intraperitoneally for 6 days/week for 8 weeks.After 8 weeks of administration,the mice were executed and eyeball blood,the liver and kidney tissues,bilateral tibia and femurs were collected.The bone morphology parameters such as bone volume fraction(BV/TV),bone trabecular thickness(Tb.Th),number of bone trabeculae(Tb.N)and bone trabecular separation(Tb.Sp)were analyzed by microcomputer tomography(Micro-CT).The degree of inflammatory cell infiltration was evaluated by H&E staining,the osteogenesis was detected by Masson staining,the expression of TNF-?,IL-10,OCN and Runx2 was detected by immunohistochemical staining,and the polarization of macrophages was evaluated by immunofluorescence staining.The levels of TNF-?,IL-1?,IL-6 and IL-10 in peripheral blood were measured by ELISA,and the liver and kidney tissue sections were stained with H&E to evaluate the injury of liver and kidney.Results:The results of Micro-CT showed that the bone volume fraction,average bone trabecular thickness and the number of bone trabeculae in Low-TF3 group and High-TF3 group respectively increased by 27.3%,20.0%and 10.9%,and 48.5%,86.7%and 33.3%.The separation of bone trabeculae decreased by 14.6%and 21.9%respectively,and the difference was statistically significant(p<0.01).The results of H&E staining showed that the number of bone trabeculae in distal femur of mice treated with TF3 was significantly higher than that of OVX group,the inflammatory reaction was alleviated,the number of inflammatory cells was significantly decreased,and the destruction of bone trabeculae was significantly improved.The degree of improvement was significantly dose-dependent.Masson staining results show that TF3 can significantly promote collagen increase and promote osteogenesis.The number of OCN and Runx2 positive cells in ovariectomized mice after TF3 treatment was significantly higher than that in OVX group,and the increase was dose-dependent.The results of immunohistochemical staining showed that the number of osteoblast-related markers in ovariectomized mice was significantly higher than that in OVX group.The expression of proinflammatory factor TNF-? decreased and the expression of anti-inflammatory factor IL-10 increased in ovariectomized mice after TF3 treatment.The results of TNF-? and IL-10,detected by immunohistochemical staining showed that the expression of pro-inflammatory factor TNF-? decreased and the expression of anti-inflammatory factor IL-10 increased in ovariectomized mice.The results of immunofluorescence staining showed that TF3 reversed macrophages from M1 type to M2 type and inhibited inflammation.The results of ELISA showed that TF3 could inhibit the expression of proinflammatory cytokines TNF-?,IL-1? and IL-6 in OVX mice,and promote the expression of anti-inflammatory cytokine IL-10 in OVX mice.The results of H&E staining in the sections of liver and kidney of each group showed that there was no significant damage to the liver and kidney of mice treated with TF3 for 8 weeks.Conclusion:TF3 can effectively inhibit the expression of proinflammatory cytokines,promote the polarization of M1 type macrophages to M2 type,reduce inflammation and reduce bone mass loss in ovariectomized mice.It is expected to become an effective drug for the prevention and treatment of osteoporosis.