| Objective:To retrospectively analyze the clinical efficacy of Gubi Zhitong Wan in the treatment of kidney-deficiency knee osteoarthritis,and provide a favorable reference for clinical research and treatment.The kidney deficiency rat model was established,and the kidney deficiency rats were given Gubi Zhitong Wan orally to study the regulation mechanism of Gubi Zhitong Wan on the knee articular cartilage of the kidney deficiency rat.At the same time,the application of transcriptomics to explore the relevant molecular mechanism of the traditional Chinese medicine theory "Kidney Governing Bones" provides an objective scientific basis for this theory.Methods:1.Collect 80 inpatients diagnosed with kidney-deficiency knee osteoarthritis in the Department of Traditional Chinese Medicine and Traumatology,Affiliated Hospital of Changchun University of Traditional Chinese Medicine from February 2019 to December2021.The patients in the control group were given the routine basic western medicine treatment,and the patients in the observation group were given the oral treatment of Gubi Zhitong Wan on the basis of the control treatment.The visual analogue scale(VAS)score,Lysholm’s function scoring(Lysholm)and Western Ontario and Mc Master Universities Osteoarthritis Index(WOMAC)score before and after treatment were analyzed to evaluate the treatment effect.2.According to the method of random number table,30 SPF 6-week-old male rats were divided into control group(n=10),kidney-deficiency group(n=10)and Gubi Zhitong Wan group(n=10).The rats in the control group were intramuscularly injected with 0.9% saline 0.3m L once a day for 22 days.Rats in kidney-deficiency group were injected with hydrocortisone suspension 2.5mg/100 g once a day for 22 days.The rats in the Gubi Zhitong Wan group were injected with hydrocortisone suspension 2.5mg/100 g into the hindlimb muscle once a day for 22 days.From the 8th day,the rats in the Gubi Zhitong Wan group were given 1.05 g/(kg· d)aqueous solution by intragastric administration,once a day for 15 consecutive days until the 22 nd day.The changes of body mass of rats were recorded during administration.After continuous administration for 22 days,the rats were anesthetized and the blood was taken from the abdominal aorta,and the rats were killed,and the cartilage tissues of the kidney and knee joint were taken,and the pathological sections were observed by hematoxylin-eosin(HE)staining.The expression levels of Col2a1,Col9a1,Col10a1,Col11a1,Acan,Comp,Hapln1,Ibsp,Wwp2,Sox9,Sox5 and Sox4 in knee joint cartilage of rats in each group were detected by real-time quantitative PCR(qRT-PCR).The levels of adreno cortico tropic hormone(ACTH),cortisol(CORT),luteinizing hormone(LH)and testosterone(T)were measured by ELISA.3.The transcriptome database of bone,cartilage,kidney,testis,thyroid,and adrenal tissue of kidney deficiency rats was established by RNA-seq high-throughput sequencing technology,and differentially expressed gene analysis and functional annotation were performed on the transcriptome database.At the same time,histological analysis,ELASA Methods and qRT-PCR detection,to explore the molecular mechanism of the "Kidney Governing Bones" theory of the interaction between multiple tissues.Results:1.Gubi Zhitong Wan can relieve the pain,stiffness and difficulty of daily activities in patients with kidney-deficiency knee osteoarthritis,improve knee joint function,and has a good effect on kidney-deficiency knee osteoarthritis.2.Compared with the control group,the serum levels of ACTH,T,LH and CORT in the kidney-deficiency group decreased significantly(P < 0.05),and the above indexes increased significantly after treatment with Gubi Zhitong Wan(P<0.05).Pathological section results of knee cartilage: in the kidney-deficiency group,the surface of the cartilage layer of the knee joint collapsed,the cartilage layer became thinner,the chondrocytes pyknotic,and the cartilage matrix decreased significantly.In the Gubi Zhitong Wan group,the cartilage layer was thickened and the surface was smooth and continuous.Compared with the kidney-deficiency group,the number of chondrocytes,cartilage matrix and homologous chondrocytes in the cartilage layer increased,and the trabecular components under the cartilage layer widened and the number of distribution increased.The results of renal pathological sections: there were no abnormal changes in renal tubules and glomeruli in the control group,and glomeruli dilatation,proximal convoluted tubule degeneration and distal convoluted tubule dilatation in the kidney-deficiency group.In Gubi Zhitong Wan group,the degree of glomerular dilatation was reduced,the functional state of renal tubule was improved,there was no obvious degeneration of proximal convoluted tubule and no obvious dilation of distal convoluted tubule.The expression of Col2a1,Col9a1,Col10a1,Col11a1,Acan,Comp,Hapln1,Ibsp,Wwp2,Sox9,Sox5,Sox4 and other genes in the kidney-deficiency group was significantly lower than that in the control group(P<0.05),and the expression of the above genes in the Gubi Zhitong Wan group was significantly higher than that in the kidney-deficiency group(P<0.05).3.In the state of kidney-deficiency,the functions of bones and cartilages are inhibited.Compared with the control group,kidney-deficiency rats exhibited decreased bone mineral density and significantly decreased expression levels of osteoblast and osteocyte markers,such as Col1a1,Ibsp,Bglap,Omd,Dcn,Dmp1,Sost,Phex,and Mepe.The expression levels of genes Pth1 r,Bmp3,Bmp8 a,Fgfr3 and Sox4 related to bone homeostasis were significantly reduced.The expression levels of chondrocyte markers including Sox9,Sox8,Sox5,Sox4,Acan,Col2a1,Col9a1,Col11a1,Hapln1,and Wwp2 were decreased in the kidney deficiency state.The expression levels of prehypertrophic chondrocyte markers,including Fgfr3,Matn1,Comp,and Runx2,were decreased in the state of kidney deficiency.In addition,4 organs(thyroid,adrenal,kidney,and testicular tissue)involved in the endocrine and genitourinary systems were also studied,and we found that their functions were also suppressed in the state of kidney deficiency.Transcriptome sequencing showed the expression levels of kidney function and testis markers(Cd28,Slc4a5,Slc38a3,Slc22a2,Slc22a12,Aqp7,Ephb1,Hmgb2,Col1a1,Cyp2a1,Ddit4,Col3a1,Igfbp4,Ccdc39,C1galt1,and Scrn1).Decreased under the influence of kidney deficiency.At the same time,we found genes with a consistent expression pattern in the adrenal gland and bone(cartilage),and genes with a consistent expression pattern in the thyroid and bone(cartilage).And multiple pathways were found by GO and KEGG enrichment analysis.Conclusion:Clinical retrospective analysis found that Gubi Zhitong Wan had a good effect on kidney-deficiency knee osteoarthritis.After animal experiments,we found that Gubi Zhitong Wan can effectively improve the pathological state of kidney deficiency in rats,and by regulating Col2a1,Col9a1,Col10a1,Col11a1,Acan,Comp,Hapln1,Ibsp,Wwp2,Sox9,Sox5,Sox4 and other genes,So as to achieve the effect of protecting cartilage.The research results of the theory of "Kidney Governing Bones" show that its action pathway involves multiple systems and organs.The "kidney" in traditional Chinese medicine does not only refer to the anatomical term "kidney",but involves the endocrine urogenital system The role of co-regulation.The homeostasis of bone and cartilage is affected in the state of kidney deficiency.The "Kidney Governing Bones" is a complex biological network changes caused by functional changes of multiple genes.This also coincides with the holistic view of traditional Chinese medicine.The human body itself is an organic whole,and the functional activities of various zang-fu organs are coordinated with each other and affect each other pathologically. |
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