Effect of Duhuo Jisheng Decoction on miRNA-17-5p Expression and PI3K/Akt/mTOR/GSDMD Signaling Pathway in a Rat Model of Knee Osteoarthritis

doi:10.21203/rs.3.rs-3109433/v1

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Effect of Duhuo Jisheng Decoction on miRNA-17-5p Expression and PI3K/Akt/mTOR/GSDMD Signaling Pathway in a Rat Model of Knee Osteoarthritis

https://doi.org/10.21203/rs.3.rs-3109433/v1

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Knee osteoarthritis (KOA) is a prevalent chronic knee joint disease characterized by cartilage degeneration and joint damage, with pain and joint deformity as the primary clinical manifestations. Emerging literature indicates that pyroptosis plays a pivotal role in the progression of KOA. Traditional Chinese medicine (TCM), especially Duhuo Jisheng Decoction has exhibited anti-inflammatory and analgesic potential in the treatment of KOA. However, it remains unclear whether it can affect pyroptosis and the underlying mechanisms. In this study, we established a rat model of KOA, and the Lequesne MG score confirmed the successful establishment of the arthritis model, demonstrating the therapeutic efficacy of Duhuo Jisheng Decoction in KOA treatment. Histopathological analysis showed a reduction in pathological damage to the synovial tissue of knee joints in Duhuo Jisheng Decoction treatment groups. Moreover, Duhuo Jisheng Decoction exhibited a regulatory effect on inflammatory factors in joint effusion. Consistent beneficial effects of Duhuo Jisheng Decoction were also observed in IL-1β induced SW1353 cells in vitro. We found that Duhuo Jisheng Decoction upregulated the expression of miR-17-5p. Both Duhuo Jisheng Decoction or overexpression of miR-17-5p can suppress the activation of PI3K/AKT/mTOR signaling pathway, thereby inhibiting the cleavage of GSDMD invlovled in pyroptosis. Together, our results suggest that Duhuo Jisheng Decoction regulates the PI3K/Akt/mTOR signaling pathway via miRNA-17-5p, thereby affecting the expression of pyroptosis key protein GSDMD. These findings contribute to our understanding of the relevant mechanisms in the treatment of KOA and provide insights into the application of TCM in managing KOA.

Knee osteoarthritis (KOA) is a prevalent chronic knee disorder characterized by cartilage degeneration, destruction, and osteophyte formation ¹. Early symptoms include joint pain and stiffness, which can progress to knee joint deformity and dysfunction, significantly affecting patients' physical and psychological well-being ^2,3. KOA primarily affects middle-aged and elderly individuals, with a 60% prevalence in patients over 50 years of age, and an increasing incidence trend with advancing age ^4,5.

Chinese medicine has a long history of treating KOA. Recognized for its effectiveness alongside very few side effects, together with its suitability for long-term KOA treatment, Chinese medicine has become a popular research topic in preventing and treating the disease. According to Chinese medicine, KOA pathogenesis involves a deficiency of origin and symptoms, with liver and kidney deficiency as the origin and wind, cold, and dampness as the symptoms and reality ⁶. Duhuo, a Chinese herbal medicine recipe, was created by Sun Simiao during the Tang Dynasty as a remedy for chronic paralysis resulting from Qi and blood deficiency in the liver and kidney, as documented in the Preparedness and Urgency of the Thousand Gold Prescriptions ⁷. Although Duhuo has demonstrated therapeutic efficacy in KOA pathogenesis, the specific molecular mechanism of its improvement in KOA remains to be explored.

Studies have shown that autophagy and apoptosis are crucial pathological processes involved in KOA cartilage lesions ^8,9. Beclin-1¹⁰, B-cell lymphoma-2 (Bcl-2) ¹¹ and cysteine aspartate-3 (Caspase-3) ¹² regulate chondrocyte autophagy and apoptosis through cartilage metabolism. While emerging literature indicates that pyroptosis plays a pivotal role in the progression of KOA. Pyroptosis is a novel form of pro-inflammatory programmed cell death distinct from apoptosis and necrosis, which can trigger the release of cytokines and activate immune cell mediators involved in inflammation. The execution of pyroptosis relies on the key component Gasdermin D (GSDMD). Cleavage of GSDMD by active caspase-1 results in the release of the N-terminal fragment of GSDMD (GSDMD-N), which forms pores in the cell membrane and facilitates the release of interleukin 1β (IL-1β). Activation of IL-1β by caspase-1 cleavage not only triggers apoptosis¹³, but also stimulates secretion of cartilage degrading enzymes such as matrix metalloproteinase (MMP) 3, MMP13, and disintegrin with thrombospondin motif and metalloproteinases-4 and − 5^14,15. This leads to the decomposition of type II collagen and proteoglycan in the extracellular matrix, exacerbating the occurrence and progression of osteoarthritis. Hence, there is a pressing demand for novel approaches to suppress pyroptosis and alleviate KOA.

Tiny RNA molecules actively participate in regulating various molecular metabolic processes in living organisms ¹⁶. Research indicates that miRNAs play a significant regulatory role in osteoarthritis pathology ¹⁷ and contribute to the development of numerous diseases by regulating autophagic activity and pyroptosis ¹⁸. Li et al. have shown that miR-155 inhibits the NLRP3/Caspase-1 pathway by targeting SMAD2, thereby inhibiting chondrocyte pyroptosis to play a role in the treatment of knee osteoarthritis¹⁹. Wang et al. also found that miR-219a-5p inhibits pyroptosis in knee osteoarthritis via inactivating the NLRP3 signaling²⁰. Chang et al. comprehensively analyzed the key genes, signaling pathways and miRNAs of human knee osteoarthritis based on bioinformatics. The expressions of miRNAs such as miR-17, miR-21, miR-155, miR-185 and miR-1 have significant changes in knee osteoarthritis, but there is a lack of research, which is worthy of further exploration²¹. Among them, miR-17‑5p is found to contribute to osteoarthritis progression by binding p62/SQSTM1²².

The phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway has been closely associated with KOA development ²³. It is well known that the PI3K/Akt/mTOR signaling pathway can regulate the proliferation, apoptosis and differentiation of various cells, and this pathway is the most important way to regulate the proliferation, apoptosis and matrix remodeling of chondrocytes²⁴. Moreover, studies have shown that the PI3K/AKT signaling pathway can be activated in multiple ways in the early stage of osteoarthritis²⁵. Therefore. in this study, we established a rat model of knee osteoarthritis to investigate the correlation between Duhuo's involvement in regulating miRNA-17-5p expression and pyroptosis via the PI3K/Akt/mTOR /GSDMD signaling pathway. We also aimed to provide insights into the application of tradition chinese medicine in ameliorating KOA.

Experimental animal grouping and handling

A total of 24 healthy male SD rats were provided by the Fourth Affiliated Hospital of Harbin Medical University, and the 24 rats were randomly divided into sham-operated group, model control group, positive control group (model rats + sodium glacial acid treatment), and unique live treatment group (model rats + unique live feeding), with 6 animals in each group.

Main reagents and instruments

Papain (917B023), L-cysteine (314B024) and paraformaldehyde (20140827) were purchased from Beijing Solaibao Technology Co; Sodium glutamate (Spironolactone, supplied by Shandong Phosphor Frita Pharmaceutical Co., Ltd.); SW1353 (human osteosarcoma cells) is a product of Proxel;Hematoxylin, eosin, Trizol and DEPC treated water were purchased from BASO; real-time fluorescence quantitative PCR instrument (Model 7300) was from ABI, USA ; tissue RNA extraction kit (Cat# 15596-026) was purchased from Invitrogen; biological spectrophotometer was purchased from Hangzhou Aosheng Instruments Ltd; electrophoresis instrument was purchased from Beijing Liuyi Biotechnology Co.

Establishment of ROA rat model and treatment

Except for the sham-operated group, all groups of rats underwent KOA rat osteotomy, and the osteotomy was performed by drug injection into the knee joint cavity (Takacs et al., 2014), Anesthetized with chloral hydrate (40 mg/kg, intraperitoneal injection), 4% papain solution (0.2 mL) was mixed with 0.03 mol/L cysteine 0.1 mL and left to stand for 0.5 h. The mixture (20 µL) was injected into the left and right knee joints of rats every other day for 3 times to establish the model. The modelling period was 2 weeks in total and the intervention was administered to each group of rats after the animals had stabilised. During the experiment, the rats were observed for their mental, dietary and activity status. 2 weeks later, the rats were considered to be successfully moulded if they showed flexion and extension dysfunction, skipping, shuffling and reduced activity on the moulded side, with a Lequesne MG score of ≥ 6. In the positive drug group, after successful membrane formation, rats were injected with a concentration of 10 mg/mL sodium glassate injection into both knee cavities at 60 µL/knee once a week for 3 weeks; in the doklam treatment group, after successful membrane formation, the drug was given by gavage using doklam parasitic soup 1 g/kg once a day, and the material was taken after 3 weeks of administration.

Cell culture and transfection

Chondrosarcoma (SW1353) cells were cultured at 37℃ in L-15 medium with 10% fetal bovine serum. The passaged SW1353 cells were digested and the number of cells was counted after centrifugal resuspension. Then, the cells were seeded at a density of 1 × 10⁶ cells into T25 culture flasks. SW1353 cells were transiently transfected with miRNA-17-5p mimic, miRNA-17-5p inhibitors, or negative controls (NC) according to the manufacturer’s instructions. After 6 hours of transfection, the medium was replaced by fresh medium with or without 10 ng/mL IL-1β. Then the cells were used for protein/RNA extraction.

International Osteoarthritis Scale score (LequesneMG)

The rats in each group were scored on the LequesneMG scale: local pain irritation response (0–3 points), gait score (0–3 points), joint mobility (0–3 points), degree of joint swelling (0–2 points), and total score range (0–11 points) before the membrane was made, after the moulding and after the intervention treatment.

Histopathological examination of the rat knee joint

Two rats in each group were randomly selected and anaesthetized with chloral hydrate (40 mg/kg, intraperitoneal injection), then the cervical vertebrae were dislocated and executed, the bilateral hindfoot joints were dissected and removed, two pieces of joint tissue were cut out from each rat, both about 0.8 g, grouped and labelled and stored in liquid nitrogen。The remaining joint tissues were rinsed, dehydrated, transparent and embedded, and then selected to be of uniform thickness and without breakage.

ELISA assay for MMP3, 1L-1β, 1L-6 and TNF-α levels

The levels of MMP3, 1L-1β, 1L-6 and TNF-α in rat myocardial tissue were determined individually by Elisa kit using 0.8g of joint tissue stored in liquid nitrogen. For details, please refer to the kit instructions.

Real-time fluorescence quantitative PCR

0.8 g of joint tissues preserved in liquid nitrogen were extracted, and the expression levels of mi-RNA-17-5p, ITGB8, JAK1, AKT and ERK genes in rat knee cartilage were measured by real-time fluorescence quantitative PCR, the tissues were cut and ground in liquid nitrogen, and the total RNA was extracted by Trizol method, and the RNA concentration was calculated with the aid of UV spectrophotometer. The samples were reverse transcribed by adding the appropriate reagents at 37 ℃ for 1 h; 85 ℃ for 5 min; and 4 ℃ for 5 min. The PCR reaction system was 2.0 µL and 9.5 µL each for cDNA template and RNase-free water, 0.5 µL each for upstream and downstream primers, and 12.5 µL of SYBRGreen Mix. 10 min pre-denaturation at 95 ℃, 95 ℃, reaction 15s at 95 ℃ and 45s at 60 ℃. The fluorescence signal was also collected and cycled 40 times, and three replicate wells were set up. Details of the primer sequences are shown in the table below.

Western blot assay

0.8 g of joint tissue preserved in liquid nitrogen was removed and the expression levels of A3R, β-Catenin, p-ERK and ERK in rat knee cartilage were measured by Western blot. The joint tissue was homogenised and then the supernatant was retained, after which the protein concentrations were determined with the aid of the BCA method. Equal amounts of tissue proteins were taken, diluted equivalently, and denatured for 10 min at 100 ℃, followed by 10% SDS (sodium dodecyl sulfate) - PAGE (polyacrylamide gel electrophoresis), with proteins transferred semi-dry at 25V for 30 min and then blocked by TBST skimmed milk (5%) for 60 min at 37 ℃.

Immunohistochemical staining

0.8 g of joint tissue stored in liquid nitrogen was removed, sections (3 µm thickness) were made, dried and baked, xylene transparent, gradient dehydrated, antigen repaired under high pressure, goat serum closed, primary antibody incubated for 1 h, then secondary antibody (1:400) was added, rinsed, shaken dry, slices were sealed with anti-fluorescence quenching sealer (containing DAPI) and finally fluorescent sections were scanned by a digital section scanner.

Statistical analysis

GraphPad Prism was used for data analysis. Statistical analysis was performed using one-way analysis of variance, followed by Fisher's PLSD test. p < 0.05 between two groups was considered to be significantly different.

Establishment and evaluation of animal arthritis model

As depicted in Table 1, the Lequesne MG score in the model group was significantly higher than that in the sham operation group (^ap < 0.05). In comparison to the model group, the Lequesne MG scores in both the positive drug treatment group and the active treatment group were lower, exhibiting significant differences (^bp < 0.05). This outcome confirmed the successful establishment of the arthritis model and suggested that active treatment can effectively address knee osteoarthritis.

Table 1

LequesneMG score
Group	n	LequesneMG
Normal	6	0.00 ± 0.00^b
KOA	6	5.17 ± 0.37^a
Hyaluronate Injection	6	1.50 ± 0.50^ab
DHJST	6	2.33 ± 0.47^ab
^ap < 0.05, compared with the Normal group; ^bp < 0.05, compared with the knee osteoarthritis (KOA) group. Duhuo Jisheng Decoction treated group (DHJST)

Effect of Duhuo Jisheng Decoction on inflammatory factors of joint effusion

As shown in Fig. <link rid="fig1">1</link>, A-1 represents normal synovial tissue in a rat's knee, while A-2 shows a rat afflicted with arthritis. The HE staining results revealed loose synovial tissue in the knee. A-3 and A-4 display slices of synovial tissue from rats treated with positive drugs and Duhuo Jisheng Decoction, respectively. It can be observed that the knee synovial tissue in both cases has recovered to varying extents. This suggests that both active and positive drug treatments can reduce the pathological damage to the knee joint's synovium and lessen the severity of joint tissue injury.

Moreover, Duhuo Jisheng Decoction also influenced the levels of IL-6, IL-1β, TNF-α, and MMP3 inflammatory factors in the synovial fluid of rats with KOA. As illustrated in Fig. 1.B, after treatment, IL-6, IL-1β, TNF-α, and MMP3 levels in both the positive drug group and the Duhuo Jisheng Decoction treated group (DHJST)-were reduced to various extents compared to the arthritis samples. Table 2 provides specific figures to further elucidate this observation. ELISA data demonstrated that both Duhuo Jisheng Decoction and positive drugs can decrease the level of inflammatory factors in joint effusion, thereby reducing the inflammatory response.

Table 2

Duhuo Jisheng Decoction on synovial fluid levels of IL-6, IL-1β, TNF-α and MMP3 in knee osteoarthritis rats
Group	n	IL-6(pg/mL)	IL-1β(pg/mL)	TNF-α(pg/mL)	MMP3(ng/mL)
Normal	3	176.37 ± 6.87^b	180.66 ± 2.89^b	117.05 ± 3.38^b	12.16 ± 0.66^b
KOA	3	313.30 ± 9.48^a	304.62 ± 5.03^a	186.19 ± 5.18^a	28.60 ± 0.67^a
Hyaluronate Injection	3	193.98 ± 3.71^ab	193.98 ± 3.71^ab	132.32 ± 2.40^ab	13.49 ± 0.99^ab
DHJST	3	239.12 ± 3.50^ab	209.96 ± 4.10^ab	143.73 ± 2.03^ab	18.46 ± 045^ab
^ap < 0.05, compared with the Normal group; ^bp < 0.05, compared with the KOA group

Duhuo Jisheng Decoction affected the expression of miRNA-17-5P, PI3K/Akt/mTOR and GSDMD proteins

ERK protein kinase is an essential molecule responsible for regulating cell growth, and the PI3K/Akt/mTOR pathway is connected to numerous other signaling pathways. The qPCR and Western blot results in Fig. 2 revealed that Duhuo Jisheng Decoction affected the expression of genes and proteins in miRNA17-5p and ERK pathways in joint tissues of a rat model with knee osteoarthritis. In comparison to the control group, the expression levels of miRNA17-5p and A3R in the KOA group were significantly decreased, but could be partially restored by Duhuo Jisheng Decoction. The expressions of β-catenin, ITGB8, and JAK1 were significantly increased and could also be partially restored by Duhuo Jisheng Decoction. Concurrently, it was discovered that the GSDMD protein in the activated pyroptosis pathway was present in knee arthritis, suggesting that the pyroptosis pathway may be involved in the pathogenesis and treatment of knee osteoarthritis.

Establishment of cell arthritis model in vitro

According to previous reports, 10 ng/mL IL-1β was used to induce in vitro chondroarthritic models. The results of the CCK8 experiment demonstrated that the secretion and expression levels of IL-6 and MMP3 were significantly increased in the IL-1β-induced chondroarthritic in vitro model, indicating the successful establishment of this model (Fig. 3. A and B). Based on this, miRNA-17-5p overexpression and knockout materials were constructed. Compared to the control group, the expression of miRNA-17-5p in the IL-1β-induced model group was significantly decreased, which was partially restored by Duhuo Jisheng Decoction. A similar effect could be achieved by applying only the active ingredient gentianine alone (Fig. 3. C).

The contents of IL-6, IL-1β, TNF-α and MMP3 in the supernatant of cells treated with gentianine were determined

The levels of IL-6, IL-1β, TNF-α, and MMP3 in the supernatant of cells treated with Duhuo Jisheng Decoction and its active component (gentianine) in Table 3 showed that the expressions of these pro-inflammatory cytokines were lower in the group after treatment. Overexpression of miRNA-17-5p approached the normal level. This suggests that miRNA-17-5p is involved in mediating the onset of arthritis, and when its expression is upregulated, it can reduce the production of pro-inflammatory cytokines and inhibit joint inflammation. The secretion of pro-inflammatory factors can also be inhibited by gentianine.

Table 3

Results of IL-6, IL-1β, TNF-α and MMP3 contents in cell supernatant(X ± S)
Group	IL-6(pg/mL)	IL-1β(pg/mL)	TNF-α(pg/mL)	MMP3(ng/mL)
NC	57.28 ± 2.65^b	29.22 ± 1.31^b	51.78 ± 1.70^b	183.21 ± 7.06^b
IL-1β	97.21 ± 1.16^a	66.76 ± 2.97^a	113.71 ± 3.81^a	267.33 ± 3.12^a
IL-1β + DHJST	61.25 ± 1.58^b	31.56 ± 5.13^b	55.14 ± 1.01^b	191.75 ± 1.80^b
IL-1β + DHJST + miRNA-17-5P(OE)	52.47 ± 0.86^b	26.39 ± 2.32^b	49.81 ± 1.70^b	177.33 ± 4.90^b
IL-1β + DHJST + miRNA-17-5P(KD)	99.62 ± 1.44^acd	69.88 ± 2.55^acd	113.50 ± 1.89^acd	273.28 ± 3.22^acd
IL-1β + Erythricine	62.13 ± 2.33^be	36.58 ± 2.88^be	48.23 ± 1.21^be	192.41 ± 5.34^be
IL-1β + Erythricine + miRNA-17-5P(OE)	52.62 ± 1.32^be	24.90 ± 3.28^be	60.09 ± 2.76^be	174.87 ± 3.99^be
IL-1β + Erythricine +miRNA-17-5P(KD)	96.06 ± 2.21^acdfg	69.01 ± 1.49^acdfg	113.09 ± 1.75^acdfg	273.60 ± 3.10^acdfg
^ap<0.05, compared with the NC; ^bp<0.05, compared with the IL-1β; ^cp<0.05, compared with the IL-1β + Duhuo Jisheng Decoction; ^dp<0.05,compared with the IL-1β + Duhuo Jisheng Decoction + miRNA-17-5P(OE); ^ep<0.05,compared with the IL-1β + Duhuo Jisheng Decoction miRNA-17-5P(KD); ^fp<0.05,compared with the IL-1β + Erythricine; ^gp<0.05,compared with the IL-1β + Erythricine + miRNA-17-5p (OE); ^hp<0.05,compared with the IL-1β + Erythricine + miRNA-17-5p (KD)

Effect of Gentidine and its active component on PI3K/AKT/mTOR/ERK signaling

Western blot results (Fig. 4) demonstrated that the overexpression of gentianine and miRNA-17-5p inhibited the phosphorylation of PI3K, AKT, mTOR, and ERK, consequently blocking the PI3K/AKT/mTOR/ERK signal transduction. This can effectively delay the development of arthritis.

IL-1β can induce the division of GSDMD, a pyrogenic protein pathway molecule

The results of Western blot revealed that IL-1β could induce the cleavage of the GSDMD pathway molecule, producing more GSDMD-N protein (Fig. 5). The cleavage of GSDMD was effectively inhibited when disulfiram was applied at concentrations of more than 10 µM. Application of polyphyllin VI at concentrations above 100 ng/mL could effectively promote the cleavage of GSDMD. The cleavage of GSDMD protein in the pyroptotic pathway could be blocked by the application of gentianine. Overexpression of miRNA17-5p can also inhibit GSDMD cleavage in the pyroptotic pathway, suggesting that miRNA17-5p is involved in the same regulatory pathway as solitary activity.

Knee osteoarthritis (KOA) is a degenerative joint disease that can cause damage to joint cartilage, subchondral bone, meniscus, and other tissues, mostly affecting middle-aged and elderly individuals. The main clinical manifestations include pain, limited activity, and joint deformity, which can severely impact patients' quality of life. Western drugs used to treat knee arthritis are mainly analgesics and cartilage protection drugs. Although they can provide quick relief, they have certain dependencies and drug resistance, making them unsuitable for long-term use.

This study utilized Duhuo Jisheng Decoction, a traditional Chinese medicine, to avoid these problems. It has dampness-removing properties, dispels wind dampness, alleviates pain, and nourishes the liver and kidneys while promoting qi and blood flow. The results confirmed that Duhuo Jisheng Decoction and its active ingredient gentianine can reduce synovial damage, joint tissue injury, and inflammation levels in a rat model of knee osteoarthritis. Additionally, it influences the expression of genes and proteins in the miRNA17-5p and ERK pathways and is involved in the pyroptotic pathway in the pathogenesis and treatment of KOA.

However, the study has some limitations. The therapeutic effect of Duhuo Jisheng Decoction and gentianine is not as strong as that of positive control drugs, and the treatment cycle is longer. Since Duhuo Jisheng Decoction is a traditional Chinese medicine preparation, it contains multiple components, and isolating a single active ingredient may not be possible. Further experiments are needed to confirm whether miRNA-17-5p is involved in the PI3K/Akt/mTOR/GSDMD signaling pathway.

In conclusion, the mechanism of Duhuo Jisheng Decoction and its active ingredient gentianine in treating knee arthritis still requires further exploration.

Ethical Approval

It has been approved by the Experimental Animal Use and Management Committee (IACUC) of Heilongjiang University of Traditional Chinese Medicine, and the approval number of the experimental animal welfare ethics review is 2021091101I hereby affirms that the study described above will be conducted with the utmost care, adhering to the highest ethical standards and guidelines for animal research. The welfare and well-being of the animals involved in the study will be given utmost priority.

Thank you for considering this request for ethical approval. Should you require any additional information or documents, please do not hesitate to contact me. I would be happy to provide any further clarification necessary.Competing interests

I declare that the authors have no competing interests as defined by BMC, or other interests that might be perceived to influence the results and/or discussion reported in this paper.

Authors' contributions

Contribution Statement: Hanbing Song and Hongpeng Liu contributed equally to this work as first authors. They were responsible for the conception and design of the study, data collection and analysis, results interpretation, and manuscript drafting. Pengfei Li and Zhigang Li participated in data collection, performed laboratory experiments, and contributed to the interpretation of the findings. Linqin He、Zonghan Tang and Qipeng Chen assisted with data analysis, interpretation of results, and critically revising the manuscript for important intellectual content. Fei Wang provided guidance and expertise in the design of the study, interpretation of data, and critically revising the manuscript. Xiaodong Li supervised the entire research project as the corresponding author. He provided substantial input in the study's conception, design, and interpretation, ensuring its scientific rigor. Xiaodong Li also critically reviewed and revised the manuscript for important intellectual content. All authors have read and approved the final version of the manuscript submitted to the Journal of Orthopaedic Surgery and Research.

Funding

Natural Science Foundation of Heilongjiang YQ2020H028
Heilongjiang research project on traditional Chinese medicine ZHY19-033
Research project of “Outstanding Innovative Talents Support Program” at Heilongjiang University of Traditional Chinese medicine 2018RCQ07

Availability of data and materials

All of the material is owned by the authors and/or no permissions are required.

Acknowledgments

This study was supported by XXX and XXX.

Disclosure

The authors declare that they have no competing interests.

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Effect of Duhuo Jisheng Decoction on miRNA-17-5p Expression and PI3K/Akt/mTOR/GSDMD Signaling Pathway in a Rat Model of Knee Osteoarthritis

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Abstract

Figures

Introduction

Materials and methods

Experimental animal grouping and handling

Main reagents and instruments

Establishment of ROA rat model and treatment

Cell culture and transfection

International Osteoarthritis Scale score (LequesneMG)

Histopathological examination of the rat knee joint

ELISA assay for MMP3, 1L-1β, 1L-6 and TNF-α levels

Real-time fluorescence quantitative PCR

Western blot assay

Immunohistochemical staining

Statistical analysis

Results

Establishment and evaluation of animal arthritis model

Effect of Duhuo Jisheng Decoction on inflammatory factors of joint effusion

Duhuo Jisheng Decoction affected the expression of miRNA-17-5P, PI3K/Akt/mTOR and GSDMD proteins

Establishment of cell arthritis model in vitro

Effect of Gentidine and its active component on PI3K/AKT/mTOR/ERK signaling

IL-1β can induce the division of GSDMD, a pyrogenic protein pathway molecule

Discussion

Declarations

References

Additional Declarations

Status:

Version 1