| Study 1:Exploration of the experience of Chinese medicine master Professor Xue Boshou in treating knee osteoarthritis based on data mining methodObjectiveTo summarize xue’s experience in treating knee osteoarthritis by combination the method of data mining technology and man-machine.MethodsThe symptom information,disease syndrome characteristics,treatments,prescriptions and characteristics of medication of Xue Boshou’s clinical medical records were analyzed,in order to systematically summarize the prescription drug law of Xue Lao’s treatment of KOA,by establishing the medical record database of Xue Boshou in treating of knee osteoarthritis,using data mining methods such as OLAP and complex network technology,and combinating the method of expert interview and man-machine.Results(1)Clinical features:The main clinical symptoms are knee joint lesions,such as knee joint pain,limited movement,swelling and pain,cold,cold pain,tingling,swelling,burning pain and stiffness.Other symptoms such as lumbago,lumbago,neck and shoulder pain,hip pain,heel pain,ankle pain and other joint symptoms and chills,insomnia,spontaneous sweating,dizziness,fatigue and other systemic symptoms.In the tongue image,the tongue is light,dark,fat and moist,and the coating on the tongue is white,white and yellow.Pulse conditions are mainly composed of deep,thin,weak,stringy,smooth and complex pulse conditions.The correlation between knee pain and low back pain was strongest.(2)syndrome characteristics:kidney deficiency and dampness syndrome has the highest frequency,followed by related syndromes of kidney deficiency such as kidney Yin and Yang deficiency syndrome,kidney deficiency and wet resistance syndrome,kidney deficiency and blood stasis syndrome,kidney deficiency and damp-heat syndrome,deficiency of kidney and obstruction of phlegm syndrome and sthenia of pathogenic factors syndrome such as Dampness and heat accumulation syndrome,cold and dampness blocking collaterals syndrome,blood stasis and water stasis syndrome,dampness and turbidness blocking syndrome,qi stagnation and blood stasis syndrome.In the relationship between syndromes,the cold-dampness blocking collateral syndrome has the strongest correlation with liver-kidney deficiency syndrome,followed by kidney deficiency and cold-dampness syndrome and blood-stasis syndrome,kidney deficiency and blood-stasis syndrome and qi-stagnation and bloodstasis syndrome,kidney Yang deficiency syndrome and kidney-deficiency and colddampness syndrome.(3)Common treatment:tonifying kidney is the highest frequency,followed by dispersing cold,warming kidney,resolving dampness,heat clearing,inducing diuresis,nourishing Yin,dredging collaterals,warming Yang,activating blood,reducing phlegm,meridian warming,liver soothing,spleen strengthening,stasis and so on.(4)The characteristics of prescription selection:a total of 96 prescriptions were selected,most of which were added or reduced by ancient prescriptions.Shenzhuo Decoction had the highest frequency,followed by Huangjing Dan,Erxian Decoction,Sini Powder,Yupingfeng powder,Zhenqi Fuzheng Soup,Huangqi Chifeng Soup,Simiao San,Simiao Yong ’an Soup,Duhuo Jisheng Soup,etc.(5)Core prescription drugs:Achyranthes bidentata,Mulberry parasitic,Eucommia ulmoides,Rhizoma officinalis,Fructus ligustri lucidi,Astragalus membranaceus,Rhizoma Drynariae,Papaya,Asarum,Angelica,Ligusticum chuanxiong Hort,dried ginger,Poria cocos,fried Atractylodes macrocephala and Gastrodia elata.Drugs commonly used compatible are Raw coix used with Phellodendron phellodendri,fried Atractylodes atractylodes and Radix clematidis,Rhizoma curculiginis used with Epimedium and Anemarrhenae,Scorpion used with Polygonum cuspidatum,Chinese yam used with Cornus officinalis,Radix scrophulariae used with Lonicera japonica,Fenugreek used with Antler tablet.Each of the other syndrome types has its core and addition and subtraction changes.(6)There are 210 commonly used herbal medicines.Common Chinese herbal medicines include Achyranthes bidentata,Poria cocos,Angelica,honey prepared glycyrrhiza,fried Atractylodes atractylodes,Mulberry parasitic,radix sileris,radix puerariae,Gastrodia elata,Astragalus membranaceus,Asarum chinense,Radix clematidis,glossy privet fruit,Phellodendron phellodendri,Papaya,Bupleurum chinense,jujube,Radix Paeoniae alba,Euphorbia officinalis,ginger,Rhizoma corydalis,dried ginger,Rhizoma corydalis,Scorpion,eucommia ulmoides,Antler tablet.The frequency of tonifying drugs was the highest,followed by heat-clearing drugs,bloodactivating and stasis-removing drugs,relieving surface drugs,invigorating water and dampness drugs,regulating qi drugs and dispelling wind and dampness drugs.The Chinese medicine taste is sweet and warm,mostly belong to the kidney,liver,spleen,lung and heart meridians.In the use of toxic Chinese medicine,Xue Boshou would use scorpion,black aconite,xianmao,bitter almond,cornus rutadiae,Tribulus tribulus,Fructus cnidii and Chinadia chinadia when necessary.ConclusionIn the treatment of knee osteoarthritis,Xue Boshou believed that kidney deficiency was the root cause of KOA,and the main pathogenesis of KOA was kidney deficiency combined with cold and dampness,blood stasis,dampness and heat.In clinical treatment of KOA,tonifying kidney is the maj or method,and the methods of dispersing cold,reducing dampness,clearing heat,enriching water,promoting blood circulation and eliminating phlegm.Study2.Effects of Bushen Zhuanggu Decoction on histomorphology and serum cytokines of cartilage and subchondral bone in KOA guinea pigsObjectiveTo clarify the therapeutic effect of Bushen Zhuanggu Decoction on KOA by observing the pathological changes of cartilage and subchondral bone in Hartley guinea pig knee osteoarthritis and the level of serum cytokines.MethodsFifty 4-month-old non-pregnant female Hartley guinea pigs were randomly divided into 5 groups,including model group,rapamycin group,TCM high-dose group,TCM medium-dose group and TCM low-dose group,with 10 pigs in each group.The traditional Chinese medicine group was given Bushen Zhuanggu decoction at the dosage of 1.4g/Kg/d by gavage,and the high,medium and low dose groups were given the clinical equivalent of 1.5 times,1 times and 0.5 times of liquid volume,respectively,and were given 4.5mL,3mL and 1.5mL by gavage.Rapamycin group was intraperitoneally injected with rapamycin solution at a dose of 0.5mg/Kd/d.All animals were sacrificed after 4 weeks of intervention,and blood,knee cartilage and knee tissue of guinea pigs were collected after the death.The contents of IL-1β,IL-6 and TNF-α in serum of guinea pigs were determined by ELISA.Tissue sections of guinea pig knee joint were made,and then HE staining was performed.The morphology of knee cartilage of guinea pig was observed under light microscope and graded by Mankin score.Micro-ct was used to observe the 3D images of subchondral bone,and the morphological indexes of subchondral bone were statistically analyzed.Results(1)Mankin score showed that compared with blank model group,the scores of rapamycin group and Chinese medicine group were lower,and the difference was significant(P<0.05).Compared with rapamycin group,there was no significant difference in the scores of TCM high-dose group(P>0.05).(2)ELISA:The levels of serum inflammatory factors IL-1β,TNF-α and IL-6 were decreased in each dose of Chinese medicine group,and the levels were increased with the decrease of Chinese medicine dose(P<0.05).Compared with rapamycin group,the concentrations of IL-1β and IL-6 in TCM high-dose group had no significant difference(P>0.05).Compared with rapamycin group,the concentration of TNF-α in each dose group was lower(P<0.01).(3)Micro-CT detection found that the number of subchondral bone trabeculae in tibia of guinea pigs in the blank model group was significantly reduced,with sparse distribution,disordered arrangement and fracture,and the trabeculae gap was significantly increased,with structural loss and lacunae in some areas.Compared with the model group,the number of subchondral bone trabecular bone in the traditional Chinese medicine group and rapamycin group was more,the distribution was denser and the texture was more orderly.The number of trabecular bones in the TCM group increased with the increase of the concentration of TCM,and the trabecular bone gradually became dense.The subchondral bone structure in the high-dose TCM group was the most complete.Statistical analysis of morphological indexes of knee subchondral bone showed that compared with the model group,the bone volume fraction(BV/TV)in the high dose group,medium dose group and rapamycin group was increased(P<0.01),and there was no significant difference among the three groups(P>0.05).In the trabecular bone thickness(Tb.Th),compared with the model group,the TCM high dose group and the TCM medium dose group significantly decreased(P<0.05).Compared with the model group,the number of trabecular bone(Tb.N)was significantly increased in the traditional Chinese medicine group(P<0.01),but no significant difference was found in the rapamycin group(P>0.05).Compared with the model group,the separation degree of trabecular bone(Tb.Sp)in the high dose group and the medium dose group was significantly lower(P<0.05),but there was no significant difference between the rapamycin group and the low dose group(P>0.05).Compared with model group,there was no significant difference in structural model index(SMI)among all groups(P>0.05).Compared with the model group,the bone mineral density(BMD)was significantly increased only in the high-dose Chinese medicine group(P<0.05),while there was no significant difference in the other groups(P>0.05).(4)Electron microscopic structure showed that chondrocytes in model group showed irregular morphology and abnormal organelle structure.Chondrocytes in rapamycin group,Chinese medicine high-dose group and medium-dose group were more complete in morphology and structure,with oval cells,continuous and complete villous protrusions in the outer layer of cells,and clear and complete organelles.Autophagosomes were observed in chondrocytes of rapamycin group and Chinese medicine high-dose group.ConclusionBushen Zhuanggu Decoction can reduce the levels of inflammatory factors IL-1β,TNF-α and IL-6 in serum,and can protect and repair cartilage and subchondral bone.Study3.Exploration of the therapeutic mechanism of Bushen Zhuanggu Decoction based on PI3K/AKT and AMPK signaling pathwaysObjectiveTo explore the molecular mechanism of Bushen Zhuanggu Decoction in regulating PI3K/AKT and AMPK signaling pathway to intervene cartilage injury in knee osteoarthritis.MethodsNinety 4-month-old clean grade non-pregnant female Hartley guinea pigs were randomly divided into 5 groups,including model group,rapamycin group,high dose Chinese medicine group,medium dose Chinese medicine group and low dose Chinese medicine group,with 18 pigs in each group,and each group was divided into 2 weeks group,4 weeks group and 6 weeks group respectively.The traditional Chinese medicine group was given 1.4g/Kg/d of Bushen Zhuanggu Decoction,and the high,medium and low dose groups were given the clinical equivalent of 1.5 times,1 times and 0.5 times of liquid volume,respectively,and were given 4.5mL,3mL and 1.5mL by gavage.Rapamycin group was intraperitoneally injected with rapamycin solution at a dose of 0.5mg/Kd/d.All the animals were sacrificed at 2 weeks,4 weeks and 6 weeks respectively.After the death,the articular cartilage and knee tissue of guinea pigs were collected.The protein expressions of PI3K,Atk,AMPK,mTOR,ULK1,Beclin1 and LC3Ⅱ in guinea pig knee cartilage were detected by Wstern blot.The gene expression levels of PI3K,Atk,mTOR,AMPK,ColⅡ and ColⅩ were detected by RT-QPCR.Results(1)Wstern blot results:At 2 weeks,compared with model group,PI3K was significantly down-regulated in high dose group(P<0.01),and ULK1 was significantly up-regulated in high dose group(P<0.01).mTOR was significantly down-regulated in high dose and medium dose groups(P<0.01,P<0.05)and AMPK,Beclinl,LC3Ⅱ/LC3Ⅰ were significantly up-regulated in high dose and medium dose groups(P<0.01).There was no significant difference in Akt among different dosage groups,but it showed a decreasing trend.At 4 weeks,compared with model group,PI3K,Atk and mTOR were significantly down-regulated in TCM high-dose group(P<0.01),and AMPK,ULK1,Beclin1 and LC3Ⅱ/LC3Ⅰ were significantly up-regulated in TCM high-dose group(P<0.01).Compared with model group,mTOR was significantly down-regulated and AMPK,ULK1,Beclin1,LC3Ⅱ/LC3Ⅰ were significantly upregulated in TCM medium dose group(P<0.05).Compared with model group,there was no significant difference in the low-dose Chinese medicine group,but the expressions of PI3K,Atk and mTOR were down-regulated,and the expressions of AMPK,ULK1,Beclinl and LC3Ⅱ/LC3Ⅰ were up-regulated.At 6 weeks,compared with the model group,PI3K and mTOR were significantly down-regulated in the mediumdose and high-dose groups(P<0.01,P<0.05),and AMPK was significantly upregulated(P<0.05).Akt showed a downward trend in the high-dose and medium-dose groups,while ULK1,Beclin1,LC3Ⅱ/LC3Ⅰ all showed an upward trend.(2)RT-qPCR results:At 2 weeks,compared with the model group,PI3K gene expression level in the high-dose group was significantly down-regulated(P<0.01),AMPK gene expression level was significantly up-regulated in the high-dose group(P<0.01).Compared with the model group,the expression level of mTOR gene in high and medium dose groups was significantly down-regulated(P<0.01).Compared with the model group,the Akt gene expression level of each dose group showed a downward trend.The COL Ⅱ gene expression level was significantly up-regulated(P<0.01)and COLX gene expression level was significantly down-regulated(P<0.01),in high and medium dose groups.At 4 weeks,compared with the model group,the expression levels of PI3K and mTOR genes in the high and medium dose groups were significantly downregulated(P<0.01),the expression levels of AMPK gene were significantly upregulated(P<0.01).The expression level of Akt gene was significantly downregulated in all dose groups(P<0.01).The COL Ⅱ gene expression level was significantly up-regulated in high and medium dose groups(P<0.01),and COLX gene expression level was significantly down-regulated in high and medium dose groups(P<0.01).At 6 weeks,compared with model group,PI3K gene expression level in highdose group was significantly down-regulated(P<0.05).The expression levels of Akt and mTOR genes were significantly down-regulated(P<0.01),and the expression levels of AMPK genes were significantly up-regulated in high and medium dose groups(P<0.01).Among different dosage groups,The COL Ⅱ gene expression level was significantly up-regulated in(P<0.05,P<0.01),and the COLX gene expression level was significantly down-regulated(P<0.01).ConclusionBushen Zhuanggu Decoction can inhibit PI3K/Akt pathway,activate AMPK pathway,inhibit mTOR and improve autophagy activity,so as to restore chondrocyte homeostasis and prevent OA. |
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