Effect Of Zishen Jiangu Decoction In Prevention And Treatment Of Osteoporosis Through Nrf2/HO-1/NF-κB Signaling Pathway

Objective1.Through clinical study,we investigated the distribution of TCM syndrome types of osteoporosis(OP)patients,so as to provide theoretical basis for effective prevention and treatment of OP;2.In vivo study,we explored the bone protective effect of Zishen Jiangu Decoction on OP rats and analyzed the regulatory role of Nrf2/HO-1/NF-κB pathway in the prevention and treatment of OP by Zishen Jiangu Decoction;3.In vitro study,we explored the effect of Zishen Jiangu Decoction on the proliferation and osteogenic activity of MC3T3-E1 cells with oxidative damage,and verified the mechanism of Nrf2/HO-1/NF-κB pathway involved in the regulation of this process.Methods1.Clinical studyOP patients were involved from March 2019 to January 2020 in the Department of osteoporosis of Shenzhen integrated traditional Chinese and Western medicine hospital.We collected the patients’date,including gender,age,BMI,previous fracture history and bone mineral density.The distribution characteristics of TCM syndrome types of OP were analyzed,and the internal relationship between each syndrome type and related pathogenic factors was explored.2.Animal experimental study(1)Therapeutic effect of Zishen Jiangu Decoction on ovariectomized OP rats72 SD rats were randomly divided into 6 groups,12 rats in each group:sham group,OVX group,ALN group,DJL group,ZJL group and GJL group.Ovariectomized rats were used to establish osteoporosis model,and drug intervention was carried out 12 weeks later,the samples were taken at 12 weeks after modeling or administration.The results were as follows:①blood was collected from abdominal aorta to detect the levels of ALP and OCN;②left femur was separated to detect bone mineral density;③micro CT scanning of tibia was performed;④the length of femur was adjusted to detect the biomechanical properties of femur at the end of ③;⑤tibia was separated to observe the histomorphological changes of proximal tibia with HE staining at the end of③.(2)Regulation of Zishen Jiangu Decoction on Nrf2/HO-1/NF-κB pathway related factors in OP rats①The levels of TNF-α,IL-6,SOD and MDA in serum were detected;②the right femur of rats in(1)was taken and preserved in liquid nitrogen;③the mRNA levels of Nrf2,HO-1,IκBα and p65 in bone tissue were detected by RT-PCR;④the protein expressions of Nrf2,HO-1,1κBα,p-IκBα,p65 and p-p65 in bone tissue were detected by Western blotting.3.Cell study(1)Effect of Zishen Jiangu Decoction on proliferation and osteogenic activity of MC3T3-E1 cells injured by HZO2After 7 days of intragastric administration,the low and high concentration serum containing Zishen Jiangu decoction were prepared.MC3T3-E1 cells were divided into blank control group(blank group),oxidative damage model group(model group),N-acetylcysteine(NAC)antioxidant damage positive control group(NAC group),low-dose Chinese medicine serum intervention group(DJL group)and high-dose Chinese medicine serum intervention group(GJL group).Except the blank group,the other groups were incubated with H2O2(final concentration is 300 umol/L)for 3h to induce oxidative stress injury.Blank group and model group were cultured in blank group drug-free serum medium,NAC group was cultured in drug-free serum medium containing NAC(1mmol/L),DJL group and GJL group were cultured in serum medium containing corresponding concentration of drug-free serum respectively.The results were as follows:(1)CCK8 method was used to detect cell proliferation at 24h,48h and 72h after incubation;(2)according to the above intervention measures,osteogenic induction medium was added to blank group,control group and NAC group,while osteogenic induction medium containing different concentrations of serum was added to DJL group and GJL group respectively,and ALP staining was performed after 7 days of incubation.(2)Regulation of Zishen Jiangu Decoction on Nrf2/HO-1/NF-κB pathway in MC3T3-E1 cells with oxidative damage induced H2O2According to(1)cell grouping and intervention measures,after incubation for 24 hours,the levels of TNF-α and IL-6 in cell supernatant were detected;the levels of MDA and SOD in cell lysate supernatant were detected;the protein expressions of Nrf2,HO-1,IκBα,p-IκBα,p65 and p-p65 in cells were detected by Western blotting.Results1.Clinical study(1)A total of 178 OP patients were included,including 75(42.4%)with liver and kidney yin deficiency,28(15.6%)with kidney yang deficiency,22(12.61%)with spleen and kidney yang deficiency,23(12.79%)with kidney deficiency and blood stasis,7(3.93%)with spleen and stomach weakness,and 23(12.67%)with qi stagnation and blood stasis;(2)In each syndrome type group,compared with liver and kidney yin deficiency type,the ratio of male and female of kidney deficiency,blood stasis type,qi stagnation and blood stasis type had significant difference(P<0.05);In terms of BMI,the BMI of patients with spleen kidney yang deficiency was significantly higher than that of OP patients with spleen stomach deficiency and liver kidney yin deficiency(P<0.05);(3)In terms of bone mineral density,the lumbar BMD of patients with liver kidney yin deficiency was significantly lower than that of other groups(P<0.05);In addition,compared with kidney yang deficiency type,spleen kidney yang deficiency type and kidney deficiency with blood stasis group,the incidence of brittle fracture of liver kidney yin deficiency type was higher.2.Animal experimental study(1)Bone protective effect of Zishen Jiangu Decoction on OP rats① BMD:compared with sham group,the BMD of femur in OVX group was significantly lower(P<0.05);compared with OVX group,the BMD of ALN group,ZJL group and GJL group was significantly higher(P<0.05);②Micro CT scan:Compared with sham group,the BV/TV,Tb.Th and TB.N values were significantly lower than that of the OVX group and the Tb.Sp values were higher.Compared with OVX group,the BV/TV,Tb.Th and TB.N values were significantly higher,while the Tb.Sp values were significantly decreased in ALN group,ZJL group and GJL group.③Biomechanical test:Compared with sham group,the bending modulus,elastic load and ultimate load of left femur in OVX group were significantly decreased(P<0.05);compared with OVX group,the biomechanical parameters of ALN group,ZJL group and GJL group were increased(P<0.05).④He staining of proximal tibia:the number of trabeculae in OVX group was rare,the structure was uneven,and the connectivity became poor;while the number of trabeculae in each drug intervention group was relatively improved,the gap became smaller,and the continuity of trabeculae was better,especially in ALN and GJL groups.⑤Serum osteogenic indexes:Compared with sham group,ALP and OCN indexes of OVX group were significantly increased(P<0.05).Compared with OVX group,ALP and OCN levels of ALN group,ZJL group and GJL group were significantly decreased(P<0.05).(2)Effect of Zishen Jiangu Decoction on Nrf2/HO-1/NF-κB signaling pathway in OP rats①Comparison of inflammatory factors and oxidative stress index levels in each group:Compared with sham group,the serum levels of IL-6,TNF-α and MDA in OVX group increased,while the level of SOD decreased significantly(P<0.05);compared with OVX group,the levels of IL-6,TNF-α and MDA decreased,and the level of SOD increased significantly,especially in ZJL and GJL groups(P<0.05).②RT-PCR detection:Compared with sham group,the mRNA levels of Nrf2,HO-1 and IκBα in OVX group were significantly decreased,and the p65 level was significantly increased(P<0.05);after intervention with traditional Chinese medicine,the mRNA levels of Nrf2,HO-1 and IκBα in ZJL group and GJL group were significantly increased,while p65 was decreased(P<0.05).③Compared with sham group,the protein expressions of Nrf2 and HO-1 in OVX group were down regulated,while p-IκBα and p-p65 were up-regulated(P<0.05);after intervention with traditional Chinese medicine,the protein levels of Nrf2 and HO-1 in ZJL group and GJL group were up-regulated,while p-IκBα and p-p65 were down regulated.3.Cell experiment(1)Protective effect of Zishen Jiangu Decoction on oxidative damage of MC3T3-E1 cells①CCK8 detection:compared with the blank group,the proliferation activity of cells in the model group at 24h,48h and 72h was significantly decreased(P<0.05),indicating the establishment of cell oxidative stress injury model;compared with the model group,the proliferation activity of cells in NAC,DJL and GJL group was significantly increased(P<0.05).② ALP staining:the degree of ALP staining in the model group was the weakest,and the number of positive cells was less;compared with the model group,the ALP positive staining in NAC group and GJL group was significantly enhanced,and the number of positive cells was increased.(2)Effect of Zishen Jiangu Decoction on Nrf2/HO-1/NF-κB pathway in MC3T3-E1 cells with oxidative damage①Detection of oxidative stress and inflammatory factors:Compared with the blank group,MDA,IL-6,TNF-α levels in the model group were significantly increased,while SOD activity was significantly decreased(P<0.05);compared with the model group,SOD activity-was increased,while MDA,IL-6,TNF-α levels were decreased in NAC,DJL and GJL group,especially in NAC group and GJL group(P<0.05).②Western blot analysis showed that compared with the blank group,the protein expressions of Nrf2 and HO-1 in the model group were down regulated,while the protein expressions of p-IκBα and p-p65 were significantly increased(P<0.05);compared with the OVX group,the protein expressions of Nrf2 and HO-1 in NAC group and GJL group were up-regulated,and the protein expressions of p-IκBα and p-p65 were down regulated(P<0.05).Conclusion1.This study found that liver and kidney yin deficiency is the most common TCM syndrome type of primary OP patients.Patients with this syndrome type have low bone mineral density and are prone to brittle fracture.Therefore,we should focus on the prevention and treatment of this type of OP in clinic.2.Animal experimental study(1)Zishen Jiangu decoction can improve bone mineral density and bone strength,protecting bone microstructure and improving the level of bone metabolism in serum of OP rats.(2)The protective effect of Zishen Jiangu Decoction on bone may be related to the anti-oxidative stress and inhibition of inflammatory reaction by regulating Nrf2/HO-1/NF-κB pathway.3.Cell experiment(1)Zishen Jiangu Decoction could improve the proliferation and osteogenic activity of MC3T3-E1 cells injured by oxidative stress.(2)Zishen Jiangu Decoction could improve the anti-oxidative stress ability of MC3T3-E1 cells and inhibit the inflammatory reaction.This mechanism may be related to its involvement in the regulation of Nrf2/HO-1/NF-κB signaling pathway.