Application method of nuciferine-containing composition for resisting rheumatoid arthritis and osteoerosion

Abstract

The invention discloses a use method of a nuciferine-containing rheumatoid arthritis bone erosion resistant composition, and relates to the field of rheumatoid arthritis bone erosion treatment.The use method comprises the steps that 1, an applicable object is determined, and the applicable object is a patient suffering from rheumatoid arthritis and having the bone erosion condition; according to the invention, the applicable object is determined to be a patient with rheumatoid arthritis and bone erosion, so that the treatment is more targeted, unnecessary use of medicines in non-target people is avoided, the utilization efficiency of medical resources is improved, and potential risks possibly brought by the medicines to non-applicable people are reduced; the nuciferine not only has remarkable anti-inflammatory activity in vitro, but also remarkably relieves the process of CIA rat arthritis in vivo, and the nuciferine relieves inflammatory response by reducing expression levels of inflammatory factors TNF-alpha, IL-1beta and IL-6 and total IgG, IgG1 and IgG2a of immune globulins in CIA rat serum.

Description

Technical Field

[0001] This invention relates to a treatment technique for bone erosion in rheumatoid arthritis, specifically to a method of using a composition containing lotus leaf alkaloids to combat bone erosion in rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic, symmetrical, polyarticular synovitis. Globally, RA has a high prevalence; statistics show that the prevalence in the adult population varies from approximately 0.5% to 1% in different regions, with women being 2-3 times more likely to be affected than men. The disease can occur at any age, but the peak incidence is between 30 and 50 years of age.

[0003] Rheumatoid arthritis (RA) not only causes common symptoms such as joint pain, swelling, and stiffness, severely impacting patients' quality of life, but also leads to joint structural damage and functional impairment. Among these, bone erosion is one of the most serious complications of RA. During disease progression, persistent synovial inflammation causes activated osteoclasts to proliferate and differentiate, gradually eroding and destroying the bone around the joint. As bone erosion worsens, the joint space narrows and becomes deformed, ultimately potentially leading to loss of joint function, causing patients to lose their ability to work or even live independently, placing a heavy burden on the individual, their family, and society.

[0004] Current treatments for bone erosion in rheumatoid arthritis (RA) have limitations. Currently, clinically used drugs for treating RA bone erosion mainly include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDs), and biologics. These drugs primarily exert their anti-inflammatory, analgesic, and antipyretic effects by inhibiting cyclooxygenase (COX) activity and reducing prostaglandin synthesis. However, these drugs only relieve symptoms and do not inhibit the progression of bone erosion. Furthermore, long-term use may cause adverse reactions such as gastrointestinal discomfort and increased cardiovascular risk. Therefore, this paper proposes a method of using a rheumatoid arthritis bone erosion-resistant composition containing lotus leaf alkaloids. Summary of the Invention

[0005] The purpose of this invention is to provide a method of using a composition containing lotus leaf alkaloids to combat bone erosion in rheumatoid arthritis, in order to solve the problem that existing drugs can only relieve symptoms and do not inhibit the progression of bone erosion, and that long-term use may cause adverse reactions such as gastrointestinal discomfort and increased cardiovascular risk.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method of using a composition containing lotus leaf alkaloids to combat bone erosion in rheumatoid arthritis, comprising:

[0007] Step 1: Determine the applicable subjects, namely patients with rheumatoid arthritis and bone erosion;

[0008] Step 2: Determine the dosage of the composition. The dosage of lotus leaf alkaloid is determined based on the severity of the patient's condition and weight. The specific dosage can be adjusted according to the doctor's assessment of the patient's individual condition.

[0009] Step 3: Determine the administration method, which includes oral administration and injection administration. Oral administration involves preparing the composition containing lotus leaf alkaloid into tablets, capsules, or granules for oral administration. Injection administration is used when the patient cannot take oral medication or urgently needs rapid onset of action. The composition containing lotus leaf alkaloid can be prepared into an injection and administered via intravenous or intramuscular injection. For intravenous injection, the lotus leaf alkaloid injection is diluted to an appropriate concentration and then slowly injected by bolus or drip. For intramuscular injection, a suitable injection site is selected.

[0010] Step 4: Determine the treatment cycle and continue treatment with the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids. During the treatment process, the patient's condition will be assessed regularly, and the treatment plan will be adjusted according to the assessment results.

[0011] Furthermore, in step three, during the administration process, the indicators for monitoring the therapeutic effect include inflammatory marker monitoring, histopathological monitoring, and cellular level monitoring. The inflammatory marker monitoring involves periodically detecting the expression levels of inflammatory factors tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and total immunoglobulins IgG, IgG1, and IgG2a in the patient's serum, and adjusting the dosage of lotus leaf alkaloids based on the detection results.

[0012] The histopathological monitoring involves using imaging examinations, such as X-rays and Micro-CT scans, to observe the improvement in the degree of bone erosion at the joints of patients and to assess changes in pathological symptoms such as synovial cell proliferation, inflammatory cell infiltration, pannus formation, and cartilage damage.

[0013] The cellular level monitoring involves extracting relevant cell samples from patients where conditions permit, detecting the differentiation degree of helper T cells 17 (Th17) and regulatory T cells (Treg), as well as the expression levels of related genes IL-17, Foxp3, RORγt, and IL-10; simultaneously detecting the expression of cell cycle-related proteins CDK2, CDK4, CyclonD1, CyclonE1 and apoptosis-related proteins Bcl-2, Bax, Caspase3, and Closed-Caspase3 in synovial fibroblasts of rheumatoid arthritis patients, as well as the activity of the PI3K / AKT signaling pathway and transcription factor Stat3, and optimizing the treatment plan based on the detection results.

[0014] Furthermore, the dosage range of lotus leaf alkaloid in the composition is as follows: in in vitro experiments, the non-toxic concentration range when acting on mouse macrophages is 10-100 μM; in in vivo experiments, when administered to a rat model of experimental rheumatoid arthritis, low-dose and high-dose groups are set up, and the specific dosage is determined according to the rat's body weight and experimental design, so as to achieve effective treatment of bone erosion in rheumatoid arthritis.

[0015] Furthermore, in an in vitro inflammation model, the composition containing lotus leaf alkaloid was applied to lipopolysaccharide-induced mouse macrophages;

[0016] The method for assessing the inflammatory effects of lipopolysaccharide-induced mouse macrophages is as follows:

[0017] The effects of different concentrations of lotus leaf alkaloids on the cell viability of RAW264.7 cells after 24 hours of treatment were detected using a CCK-8 assay to determine the range of concentrations with no toxicity.

[0018] The effect of lotus leaf alkaloids on LPS-induced NO release in RAW264.7 cells within a non-toxic concentration range was detected using the Griess method.

[0019] The effects of lotus leaf alkaloids on the mRNA expression levels of tumor necrosis factor, interleukin-1β, interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2, and vascular endothelial growth factor in LPS-induced RAW264.7 cells were detected by RT-qPCR.

[0020] Furthermore, for the in vivo animal experimental model, female Wistar rats weighing 120±10g were randomly divided into a normal group, a model group, a low-dose lotus leaf alkaloid group, a high-dose lotus leaf alkaloid group, and a positive drug group, with 6 rats in each group. Except for the normal control group, type II collagen-induced arthritis models were established in the other groups of rats, and the combination containing lotus leaf alkaloid was administered according to the set dosage. The specific operation steps are as follows:

[0021] Step 1: During the experiment, the changes in paw volume of rats in each group were measured every 7 days, and arthritis scores were calculated.

[0022] Step 2: After the last administration, animal samples were collected, and the levels of TNF-α, IL-1β, IL-6, IgG, IgG1, and IgG2a in the serum of rats in each group were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of TNF-α, IL-1β, IL-6, and VEGF in the knee joint tissue of rats were also detected.

[0023] Step 3: Observe the histopathological changes in the ankle joint of rats in each group by hematoxylin-eosin (H&E) staining and safranin-fast green staining;

[0024] Step 4: Use X-rays to assess the degree of ankle joint injury and perform imaging scoring, and use Micro-CT scans to scan the rat ankle joint and perform bone parameter analysis.

[0025] Furthermore, when determining the dosage of lotus leaf alkaloids, the patient's gene testing results are taken into account. For patients with specific gene polymorphisms, the dosage of lotus leaf alkaloids is adjusted based on the correlation studies between genes and drug metabolism, efficacy, and adverse reactions, in order to improve the therapeutic effect and reduce the occurrence of adverse reactions.

[0026] Furthermore, the cellular-level monitoring method employs single-cell sequencing technology to perform single-cell-level gene expression analysis on extracted patient-related cell samples. This allows for a more precise understanding of cellular heterogeneity and the mechanism of action of lotus leaf alkaloids on different cell subpopulations, providing a more detailed basis for the development of personalized treatment plans.

[0027] Furthermore, in the in vitro experimental evaluation process, the evaluation method adopts an extracellular matrix stiffness simulation system to simulate the extracellular matrix stiffness environment of different pathological stages in the joints of rheumatoid arthritis patients, and to study the effect of lotus leaf alkaloid under different stiffness environments on macrophage inflammatory response, so as to more realistically reflect the in vivo pathological condition and provide more in-depth data support for the study of the mechanism of action of lotus leaf alkaloid.

[0028] Compared with the prior art, the present invention provides a method of using a composition containing lotus leaf alkaloids to combat bone erosion in rheumatoid arthritis.

[0029] This invention is specifically designed for patients with rheumatoid arthritis and bone erosion, making treatment more targeted, avoiding unnecessary use of drugs in non-target populations, improving the efficiency of medical resource utilization, and reducing potential risks that drugs may pose to non-target populations.

[0030] The dosage of lotus leaf alkaloid is determined based on the severity of the patient's condition and weight, and can be adjusted according to the doctor's assessment of the individual patient's condition. This individualized dosage determination method fully considers the differences between patients, can better balance the therapeutic effect and safety, and improve the accuracy of treatment. At the same time, combined with the non-toxic concentration range of in vitro experiments and the low and high dose groups of in vivo experiments, it provides a scientific basis for clinical dosage selection and ensures that the dosage is within a safe and effective range.

[0031] It offers both oral and injectable administration methods. Oral administration involves formulating the composition into tablets, capsules, or granules for convenient daily use and improved patient adherence. Injectable administration is suitable for patients who cannot take oral medication or require rapid onset of action. The composition can be formulated into an injectable form for intravenous or intramuscular injection. For intravenous injection, it is diluted to an appropriate concentration and then slowly injected or dripped. For intramuscular injection, an appropriate injection site is selected. This diverse administration method can meet the treatment needs of different patients and ensure timely and effective drug treatment in various situations.

[0032] In summary, lotus leaf alkaloids not only exhibit significant anti-inflammatory activity in vitro, but also significantly alleviate the progression of arthritis in CIA rats in vivo. Lotus leaf alkaloids reduce the expression levels of inflammatory factors TNF-α, IL-1β, IL-6 and total immunoglobulins IgG, IgG1, and IgG2a in the serum of CIA rats. In addition, lotus leaf alkaloids significantly improved the histopathological changes in CIA rats, including inhibiting inflammatory cell infiltration, synovial hyperplasia, pannus formation, and cartilage damage. Lotus leaf alkaloids can significantly improve the degree of bone destruction in CIA rats.

[0033] Lotus leaf alkaloids can inhibit RANKL-induced osteoclast differentiation in vitro, and can also inhibit the proliferation, migration and invasion of RA-derived FLS cells. The inhibition of FLS proliferation by lotus leaf alkaloids may be related to the inhibition of the PI3K / AKT signaling pathway.

[0034] The regulation of the expression of apoptosis-related proteins Bcl-2, Bax, Caspase3, Cleaved-Caspase3 and cell cycle-related proteins CDK2, CDK4, Cyclin D1, and Cyclin E1 induces apoptosis and cell cycle arrest in FLS cells. The inhibition of synovial fibroblast invasion by lotus leaf alkaloids may be related to the inhibition of Stat3 phosphorylation at Tyr705. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0036] Figure 1 This is a schematic diagram of the steps provided in an embodiment of the present invention. Detailed Implementation

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0038] As attached Figure 1 As shown, a method of using a rheumatoid arthritis-resistant bone erosion composition containing lotus leaf alkaloids includes:

[0039] Step 1: Determine the applicable subjects, namely patients with rheumatoid arthritis and bone erosion;

[0040] Step 2: Determine the dosage of the composition. The dosage of lotus leaf alkaloid is determined based on the severity of the patient's condition and weight. The specific dosage can be adjusted according to the doctor's assessment of the patient's individual condition.

[0041] Step 3: Determine the administration method, which includes oral administration and injection administration. Oral administration involves preparing the composition containing lotus leaf alkaloid into tablets, capsules, or granules for oral administration. Injection administration is used when the patient cannot take oral medication or urgently needs rapid onset of action. The composition containing lotus leaf alkaloid can be prepared into an injection and administered via intravenous or intramuscular injection. For intravenous injection, the lotus leaf alkaloid injection is diluted to an appropriate concentration and then slowly injected by bolus or drip. For intramuscular injection, a suitable injection site is selected.

[0042] Step 4: Determine the treatment cycle, continue to use the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloid for treatment, and regularly assess the patient's condition during the treatment process, and adjust the treatment plan according to the assessment results;

[0043] Furthermore, in step three, during the administration process, the indicators for monitoring the therapeutic effect include inflammatory marker monitoring, histopathological monitoring, and cellular level monitoring. The inflammatory marker monitoring involves periodically detecting the expression levels of inflammatory factors tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and total immunoglobulins IgG, IgG1, and IgG2a in the patient's serum, and adjusting the dosage of lotus leaf alkaloids based on the detection results.

[0044] The histopathological monitoring involves using imaging examinations, such as X-rays and Micro-CT scans, to observe the improvement in the degree of bone erosion at the joints of patients and to assess changes in pathological symptoms such as synovial cell proliferation, inflammatory cell infiltration, pannus formation, and cartilage damage.

[0045] The cellular level monitoring involves extracting relevant cell samples from patients where conditions permit, detecting the differentiation degree of helper T cells 17 (Th17) and regulatory T cells (Treg), as well as the expression levels of related genes IL-17, Foxp3, RORγt, and IL-10; simultaneously detecting the expression of cell cycle-related proteins CDK2, CDK4, CyclonD1, CyclonE1 and apoptosis-related proteins Bcl-2, Bax, Caspase3, and Closed-Caspase3 in synovial fibroblasts of rheumatoid arthritis patients, as well as the activity of the PI3K / AKT signaling pathway and transcription factor Stat3, and optimizing the treatment plan based on the detection results.

[0046] Furthermore, the dosage range of lotus leaf alkaloid in the composition is as follows: in in vitro experiments, the non-toxic concentration range when acting on mouse macrophages is 10-100 μM; in in vivo experiments, when administered to a rat model of experimental rheumatoid arthritis, a low-dose group and a high-dose group are set up, and the specific dosage is determined according to the rat's body weight and experimental design, so as to achieve effective treatment of bone erosion in rheumatoid arthritis.

[0047] Furthermore, in an in vitro inflammation model, the composition containing lotus leaf alkaloid was applied to lipopolysaccharide-induced mouse macrophages;

[0048] The method for assessing the inflammatory effects of lipopolysaccharide-induced mouse macrophages is as follows:

[0049] The effects of different concentrations of lotus leaf alkaloids on the cell viability of RAW264.7 cells after 24 hours of treatment were detected using a CCK-8 assay to determine the range of concentrations with no toxicity.

[0050] The effect of lotus leaf alkaloids on LPS-induced NO release in RAW264.7 cells within a non-toxic concentration range was detected using the Griess method.

[0051] The effects of lotus leaf alkaloid on the expression levels of tumor necrosis factor, interleukin-1β, interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2, and vascular endothelial growth factor mRNA in LPS-induced RAW264.7 cells were detected by RT-qPCR.

[0052] Furthermore, for the in vivo animal experimental model, female Wistar rats weighing 120±10g were randomly divided into a normal group, a model group, a low-dose lotus leaf alkaloid group, a high-dose lotus leaf alkaloid group, and a positive drug group, with 6 rats in each group. Except for the normal control group, type II collagen-induced arthritis models were established in the other groups of rats, and the combination containing lotus leaf alkaloid was administered according to the set dosage. The specific operation steps are as follows:

[0053] Step 1: During the experiment, the changes in paw volume of rats in each group were measured every 7 days, and arthritis scores were calculated.

[0054] Step 2: After the last administration, animal samples were collected, and the levels of TNF-α, IL-1β, IL-6, IgG, IgG1, and IgG2a in the serum of rats in each group were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of TNF-α, IL-1β, IL-6, and VEGF in the knee joint tissue of rats were also detected.

[0055] Step 3: Observe the histopathological changes in the ankle joint of rats in each group by hematoxylin-eosin (H&E) staining and safranin-fast green staining;

[0056] Step 4: Use X-rays to assess the degree of ankle joint injury and perform imaging scoring; use Micro-CT to scan the rat ankle joint and perform bone parameter analysis.

[0057] Furthermore, when determining the dosage of lotus leaf alkaloids, the patient's gene testing results are taken into account. For patients with specific gene polymorphisms, the dosage of lotus leaf alkaloids is adjusted based on the correlation studies between genes and drug metabolism, efficacy and adverse reactions, in order to improve the therapeutic effect and reduce the occurrence of adverse reactions.

[0058] Furthermore, the cell-level monitoring method employs single-cell sequencing technology to perform single-cell-level gene expression analysis on extracted patient-related cell samples, thereby gaining a more precise understanding of cell heterogeneity and the mechanism of action of lotus leaf alkaloids on different cell subpopulations, and providing a more detailed basis for the formulation of personalized treatment plans.

[0059] Furthermore, in the in vitro experimental evaluation process, the evaluation method adopts an extracellular matrix stiffness simulation system to simulate the extracellular matrix stiffness environment of the joints of rheumatoid arthritis patients at different pathological stages, and to study the effect of lotus leaf alkaloid on macrophage inflammatory response under different stiffness environments, so as to more realistically reflect the in vivo pathological condition and provide more in-depth data support for the study of the mechanism of action of lotus leaf alkaloid.

[0060] Working principle: This method is based on the pathogenesis of bone erosion in rheumatoid arthritis (RA) and the pharmacological properties of lotus leaf alkaloids. It constructs a comprehensive treatment plan. RA bone erosion is mainly caused by persistent synovial inflammation, which activates osteoclasts and leads to the destruction of periarticular bone. Lotus leaf alkaloids have a variety of biological activities such as anti-inflammation and regulation of bone metabolism. By rationally using a composition containing lotus leaf alkaloids, the progression of RA bone erosion is intervened at multiple stages to achieve the purpose of treating the disease, improving patients' symptoms and quality of life.

[0061] Principle of determining the target population: Clearly defining the target population for treatment is the foundation for effective treatment. Patients with rheumatoid arthritis and bone erosion are the target population for this composition. RA is an autoimmune disease, and bone erosion is a serious complication that can lead to joint structural damage and functional impairment. Only by using a composition containing lotus leaf alkaloid for these patients can its anti-RA bone erosion effect be fully utilized, avoiding waste of resources and potential adverse effects on unsuitable populations.

[0062] The principle of determining dosage based on the severity of the condition and weight: The severity of the condition reflects the intensity of the inflammatory response and the degree of bone erosion in the patient's body. The more severe the condition, the larger the drug dose may be required to effectively inhibit inflammation and bone destruction. Weight is also an important factor because the distribution and metabolism of drugs in the body are related to weight. Generally speaking, patients with a larger body weight may need a higher dose of drug to achieve an effective therapeutic concentration.

[0063] The principle of adjusting dosage based on doctor's assessment: Each patient's specific situation is different. In addition to the condition and weight, it may also be affected by a variety of factors such as age, gender, comorbidities, and drug tolerance. Through a comprehensive assessment, including physical examination, laboratory tests, and imaging examinations, doctors can more accurately understand the patient's individual condition, thereby making personalized adjustments to the dosage of lotus leaf alkaloids to improve the safety and effectiveness of treatment.

[0064] In vitro experiments: In mouse macrophage experiments, the cell viability of RAW264.7 cells after 24 hours of treatment with different concentrations of lotus leaf alkaloids was detected by CCK-8 assay. The non-toxic concentration range was determined to be 10-100 μM. This is because macrophages play a key role in the inflammatory response of RA. Lotus leaf alkaloids are non-toxic to macrophages within this concentration range, while also exerting anti-inflammatory effects, providing a preliminary dosage reference for in vivo drug administration.

[0065] In vivo experiments: In a rat model of experimental rheumatoid arthritis, low-dose and high-dose groups were set up. The specific dosage was determined according to the rat's body weight and experimental design to achieve effective treatment of bone erosion in rheumatoid arthritis. Animal experiments can simulate the complex environment in the human body and more intuitively observe the absorption, distribution, metabolism and excretion of lotus leaf alkaloid in the body, as well as its therapeutic effect on bone erosion in RA, providing a more reliable basis for determining the dosage for clinical use.

[0066] The principle of adjusting dosage based on gene testing results: A patient's gene polymorphism can affect drug metabolism, efficacy, and adverse reactions. For example, certain gene mutations may lead to changes in the activity of drug-metabolizing enzymes, thereby affecting the drug's concentration and duration of action in the body. By conducting gene testing, we can understand whether a patient has specific gene polymorphisms, and adjust the dosage of lotus leaf alkaloids based on the correlation between genes and drug metabolism, efficacy, and adverse reactions. This can achieve individualized and precise treatment, improve treatment effectiveness, and reduce the occurrence of adverse reactions.

[0067] Oral administration principle: The composition containing lotus leaf alkaloid is made into oral preparations such as tablets, capsules or granules, which are convenient for patients to take. After oral administration, the drug is absorbed through the gastrointestinal tract and enters the blood circulation, and is distributed to all tissues and organs of the body, including joints. This method of administration is simple to operate and has a high patient compliance rate. It is suitable for patients with relatively stable conditions who are able to eat normally.

[0068] Intravenous injection: Used when patients cannot take oral medication or urgently need rapid effects. After diluting the lotus leaf alkaloid injection to an appropriate concentration, it is slowly injected or dripped in. The drug can directly enter the blood circulation, quickly reach an effective concentration, and exert its therapeutic effect. It is suitable for patients with severe conditions who urgently need to control inflammation and relieve symptoms.

[0069] Intramuscular injection: The drug is absorbed from the muscle tissue into the bloodstream by a suitable injection site. The effect is relatively slow but the duration is longer. Intramuscular injection is a feasible option when the patient cannot take the drug orally and does not need to reach a high blood concentration immediately.

[0070] The principle behind determining the treatment cycle: Rheumatoid arthritis (RA) bone erosion is a chronic, progressive disease that requires continuous treatment with a rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids to maintain an effective drug concentration in the body and continuously suppress inflammatory responses and bone destruction. During treatment, patients' conditions are regularly assessed, including clinical symptoms, signs, laboratory tests, and imaging examinations. Treatment plans are adjusted based on the assessment results. If the condition is controlled, the drug dosage may be adjusted appropriately or the dosing interval extended; if the condition worsens, the drug dosage may need to be increased or the treatment plan changed to ensure the effectiveness and safety of the treatment.

[0071] The principle of monitoring inflammatory markers for treatment efficacy: Regularly detect the expression levels of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and total immunoglobulins IgG, IgG1, and IgG2a in the patient's serum. These inflammatory factors play an important role in the inflammatory response of RA, and changes in their expression levels can reflect the degree of inflammation in the body. The dosage of lotus leaf alkaloid is adjusted according to the test results. If the level of inflammatory factors continues to rise, it may be necessary to increase the drug dosage to enhance the anti-inflammatory effect; if the level of inflammatory factors decreases significantly, it may be considered to appropriately reduce the drug dosage to avoid adverse reactions caused by drug overdose.

[0072] The principle of histopathological monitoring: Through imaging examinations, such as X-rays and micro-CT scans, the improvement of bone erosion in the joints of patients is observed, and changes in pathological symptoms such as synovial cell proliferation, inflammatory cell infiltration, pannus formation, and cartilage damage are assessed. X-rays can show overall changes in joint structure, such as joint space narrowing and bone destruction; micro-CT scans have higher resolution and can more clearly observe changes in bone microstructure. These imaging examination results can intuitively reflect the therapeutic effect of lotus leaf alkaloids on RA bone erosion, providing an important basis for adjusting the treatment plan.

[0073] Routine cellular level monitoring: For patients with suitable conditions, relevant cell samples are extracted to detect the differentiation degree of helper T cells 17 (Th17) and regulatory T cells (Treg), as well as the expression levels of related genes IL-17, Foxp3, RORγt, and IL-10. Th17 cells and Treg cells play opposite roles in the immune regulation of RA; Th17 cells promote the inflammatory response, while Treg cells suppress it. Detecting the expression levels of these cells and genes can reveal the immune homeostasis in patients and assess the regulatory effect of lotus leaf alkaloids on the immune system. Simultaneously, the expression of cell cycle-related proteins CDK2, CDK4, CyclonD1, and CyclonE1, and apoptosis-related proteins Bcl-2, Bax, Caspase3, and Cleared-Caspase3 in synovial fibroblasts of rheumatoid arthritis patients, as well as PI... The activity of the 3K / AKT signaling pathway and transcription factor Stat3, these indicators are closely related to cell proliferation, apoptosis and signal transduction, reflecting the effect of lotus leaf alkaloids on the biological behavior of synovial fibroblasts. Based on the detection results, the treatment plan can be optimized to improve the targeting of the treatment.

[0074] Single-cell sequencing technology was used to perform gene expression analysis at the single-cell level on extracted patient-related cell samples. Traditional methods involve mixing and analyzing a large number of cells, which may mask the heterogeneity between cells. Single-cell sequencing technology can more accurately understand cell heterogeneity and the mechanism of action of lotus leaf alkaloid on different cell subpopulations, discover new therapeutic targets and biomarkers, and provide more detailed evidence for the development of personalized treatment plans.

[0075] Lipopolysaccharide-induced mouse macrophage inflammation model CCK-8 assay for cell viability: The effect of different concentrations of lotus leaf alkaloids on the cell viability of RAW264.7 cells after 24 h of treatment was detected using CCK-8 assay to determine the non-toxic concentration range. This was to ensure that when studying the anti-inflammatory effect of lotus leaf alkaloids, the drug concentration would not be too high and cause toxicity to the cells, thus affecting the accuracy of the experimental results.

[0076] The effect of lotus leaf alkaloid on LPS-induced NO release in RAW264.7 cells within a non-toxic concentration range was detected using the Griess method. NO is an important inflammatory mediator that plays a crucial role in the inflammatory response of RA. Lotus leaf alkaloid can inhibit LPS-induced NO release from macrophages, indicating that it has an anti-inflammatory effect.

[0077] RT-qPCR was used to detect the effects of lotus leaf alkaloids on the mRNA expression levels of tumor necrosis factor, interleukin-1β, interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2, and vascular endothelial growth factor in LPS-induced RAW264.7 cells. These factors are all closely related to the inflammatory response. By detecting their mRNA expression levels, we can gain a deeper understanding of the anti-inflammatory mechanism of lotus leaf alkaloids at the molecular level.

[0078] The principle of the extracellular matrix stiffness simulation system is as follows: The extracellular matrix stiffness simulation system is used to simulate the extracellular matrix stiffness environment of the joints of patients with rheumatoid arthritis at different pathological stages. The effect of lotus leaf alkaloid under different stiffness environments on the inflammatory response of macrophages is studied. During the development of RA, the stiffness of the extracellular matrix in the joints will change. This change will affect the biological behavior of cells and the inflammatory response. By simulating different stiffness environments, the pathological condition in vivo can be more realistically reflected, providing more in-depth data support for the study of the mechanism of action of lotus leaf alkaloid and helping to develop more effective treatment strategies.

[0079] Measurement of paw volume changes and arthritis scores: During the experiment, paw volume changes in each group of rats were measured every 7 days, and arthritis scores were calculated. Changes in paw volume can reflect the degree of joint swelling and are one of the important indicators for assessing RA. The arthritis score comprehensively considers multiple aspects such as joint swelling, redness, and deformity, and more comprehensively assesses the severity of the disease. By regularly monitoring these indicators, the control effect of lotus leaf alkaloids on RA can be observed.

[0080] After the last administration, animal samples were collected, and the levels of TNF-α, IL-1β, IL-6, IgG, IgG1, and IgG2a in the serum of rats in each group were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of TNF-α, IL-1β, IL-6, and VEGF in the knee joint tissue of rats were also detected. These indicators can reflect the inflammatory response and immune status in the body. By comparing the differences between different groups, the anti-inflammatory and immunomodulatory effects of lotus leaf alkaloids were evaluated.

[0081] The histopathological changes of the ankle joint of rats in each group were observed by hematoxylin-eosin (H&E) staining and safranin-fast green staining. H&E staining can show the morphology and structure of cells and tissues, while safranin-fast green staining can observe changes in cartilage and bone. These staining results can intuitively reflect the repair effect of lotus leaf alkaloid on joint tissue pathological damage.

[0082] The extent of ankle joint injury was assessed and scored using X-rays, and the ankle joint of rats was scanned using Micro-CT and bone parameters were analyzed. X-ray and Micro-CT scans can provide detailed information on joint structure, such as bone erosion and joint space narrowing. Through imaging scoring and bone parameter analysis, the therapeutic effect of lotus leaf alkaloid on bone erosion can be quantitatively evaluated, providing a reliable basis for clinical application.

[0083] The beneficial effects of the present invention are as follows: the present invention is specifically applicable to patients with rheumatoid arthritis and bone erosion, making the treatment more targeted, avoiding unnecessary use of drugs in non-target populations, improving the utilization efficiency of medical resources, and reducing the potential risks that drugs may bring to non-applicable populations.

[0084] The dosage of lotus leaf alkaloid is determined based on the severity of the patient's condition and weight, and can be adjusted according to the doctor's assessment of the individual patient's condition. This individualized dosage determination method fully considers the differences between patients, can better balance the therapeutic effect and safety, and improve the accuracy of treatment. At the same time, combined with the non-toxic concentration range of in vitro experiments and the low and high dose groups of in vivo experiments, it provides a scientific basis for clinical dosage selection and ensures that the dosage is within a safe and effective range.

[0085] It offers both oral and injectable administration methods. Oral administration involves formulating the composition into tablets, capsules, or granules for convenient daily use and improved patient adherence. Injectable administration is suitable for patients who cannot take oral medication or require rapid onset of action. The composition can be formulated into an injectable form for intravenous or intramuscular injection. For intravenous injection, it is diluted to an appropriate concentration and then slowly injected or dripped. For intramuscular injection, an appropriate injection site is selected. This diverse administration method can meet the treatment needs of different patients and ensure timely and effective drug treatment in various situations.

[0086] During drug administration, the therapeutic effect was monitored from three dimensions: inflammatory markers, histopathology, and cellular levels. Inflammatory marker monitoring regularly measured the expression levels of various inflammatory factors and immunoglobulins in the patient's serum, enabling timely understanding of changes in the inflammatory response and providing a basis for adjusting the dosage of lotus leaf alkaloid. Histopathological monitoring used imaging examinations to observe the improvement in bone erosion at the joint sites and assess changes in various pathological symptoms, directly reflecting the drug's impact on joint tissues. For patients where conditions permitted, cellular monitoring extracted relevant cell samples to detect various cell differentiation degrees, gene expression levels, and related protein expression and signaling pathway activity, providing a deeper understanding of the drug's mechanism of action at the cellular and molecular levels and offering a more comprehensive basis for optimizing treatment plans.

[0087] Using single-cell sequencing technology for cellular-level monitoring enables gene expression analysis of extracted patient-related cell samples at the single-cell level, providing a more precise understanding of cellular heterogeneity and the mechanism of action of lotus leaf alkaloids on different cell subpopulations. This provides a more detailed basis for the development of personalized treatment plans and helps to further improve treatment outcomes.

[0088] In an in vitro inflammation model, the composition was applied to lipopolysaccharide-induced mouse macrophages. The inflammatory effects were assessed using multiple detection methods. Cell viability was determined using CCK-8 assay to define the non-toxic concentration range, NO release was detected using the Griess method, and the mRNA expression levels of multiple genes were detected using RT-qPCR. This comprehensive study aimed to understand the inhibitory effect and mechanism of lotus leaf alkaloids on inflammatory responses in vitro, providing fundamental data for subsequent in vivo experiments and clinical applications. Simultaneously, an extracellular matrix stiffness simulation system was used to simulate the extracellular matrix stiffness environment at different pathological stages in the joints of rheumatoid arthritis patients. The effects of lotus leaf alkaloids under different stiffness environments on macrophage inflammatory responses were investigated, more realistically reflecting the in vivo pathological condition and providing more in-depth data support for the study of the mechanism of action of lotus leaf alkaloids.

[0089] An in vivo animal model was constructed. Female Wistar rats were randomly divided into groups to establish a type II collagen-induced arthritis model. A composition containing lotus leaf alkaloid was administered according to a set dose. The changes in rat paw volume, arthritis scores, serum levels of various inflammatory factors and immunoglobulins, and mRNA levels of related genes in knee joint tissues were detected. The pathological changes in ankle joint tissues were observed. The degree of ankle joint injury was assessed using X-rays and imaging scores were performed. Bone parameters were analyzed using Micro-CT scanning. These and other experimental procedures were used to comprehensively evaluate the therapeutic effect of lotus leaf alkaloid on bone erosion in rheumatoid arthritis in vivo, providing reliable experimental evidence for clinical application.

[0090] When determining the dosage of lotus leaf alkaloids, the patient's genetic testing results are taken into account. For patients with specific gene polymorphisms, the dosage of lotus leaf alkaloids is adjusted based on the correlation studies between genes and drug metabolism, efficacy and adverse reactions. This personalized treatment approach can fully consider the impact of the patient's genetic characteristics on drug treatment, improve treatment efficacy, reduce the occurrence of adverse reactions, and make treatment more precise, safe and effective.

[0091] The treatment involves continuous use of a rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids, with regular assessments of the patient's condition during treatment and adjustments to the treatment plan based on the assessment results. This dynamic treatment management approach ensures the effectiveness and continuity of treatment, allowing for timely adjustments to the treatment strategy based on changes in the patient's condition, thereby improving the patient's recovery rate and quality of life.

[0092] In summary, lotus leaf alkaloids not only exhibit significant anti-inflammatory activity in vitro, but also significantly alleviate the progression of arthritis in CIA rats in vivo. Lotus leaf alkaloids reduce the expression levels of inflammatory factors TNF-α, IL-1β, IL-6 and total immunoglobulins IgG, IgG1, and IgG2a in the serum of CIA rats. In addition, lotus leaf alkaloids significantly improved the histopathological changes in CIA rats, including inhibiting inflammatory cell infiltration, synovial hyperplasia, pannus formation, and cartilage damage. Lotus leaf alkaloids can significantly improve the degree of bone destruction in CIA rats.

[0093] Lotus leaf alkaloids can inhibit RANKL-induced osteoclast differentiation in vitro, and can also inhibit the proliferation, migration and invasion of RA-derived FLS cells. The inhibition of FLS proliferation by lotus leaf alkaloids may be related to the inhibition of the PI3K / AKT signaling pathway.

[0094] The regulation of the expression of apoptosis-related proteins Bcl-2, Bax, Caspase3, Cleaved-Caspase3 and cell cycle-related proteins CDK2, CDK4, Cyclin D1, and Cyclin E1 induces apoptosis and cell cycle arrest in FLS cells. The inhibition of synovial fibroblast invasion by lotus leaf alkaloids may be related to the inhibition of Stat3 phosphorylation at Tyr705.

[0095] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method of using a composition containing lotus leaf alkaloids for combating bone erosion in rheumatoid arthritis, characterized in that, include: Step 1: Determine the applicable subjects, namely patients with rheumatoid arthritis and bone erosion; Step 2: Determine the dosage of the composition, based on the severity of the patient's condition and weight; Step 3: Determine the administration method, which includes oral administration and injection administration. Oral administration involves preparing the composition containing lotus leaf alkaloid into tablets, capsules, or granules for oral administration. Injection administration is used when the patient cannot take oral medication or urgently needs rapid onset of action. The composition containing lotus leaf alkaloid can be prepared into an injection and administered via intravenous or intramuscular injection. Step 4: Determine the treatment cycle and continue treatment with the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids. During the treatment process, the patient's condition will be assessed regularly, and the treatment plan will be adjusted according to the assessment results.

2. The method of using the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids according to claim 1, characterized in that, In step three, during the administration of medication, the indicators for monitoring the therapeutic effect include inflammatory marker monitoring, histopathological monitoring, and cellular level monitoring.

3. The method of using the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids according to claim 1, characterized in that, The dosage range of lotus leaf alkaloid in the composition is as follows: in in vitro experiments, the non-toxic concentration range when acting on mouse macrophages is 10-100 μM; in in vivo experiments, when administered to a rat experimental rheumatoid arthritis model, low-dose and high-dose groups are set up.

4. The method of using the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids according to claim 3, characterized in that, For the in vitro inflammation model, the composition containing lotus leaf alkaloid was applied to lipopolysaccharide-induced mouse macrophages; The method for assessing the inflammatory effects of lipopolysaccharide-induced mouse macrophages is as follows: The effects of different concentrations of lotus leaf alkaloids on the cell viability of RAW264.7 cells after 24 hours of treatment were detected using a CCK-8 assay to determine the range of concentrations with no toxicity. The effect of lotus leaf alkaloids on LPS-induced NO release in RAW264.7 cells within a non-toxic concentration range was detected using the Griess method. The effects of lotus leaf alkaloids on the mRNA expression levels of tumor necrosis factor, interleukin-1β, interleukin-6, inducible nitric oxide synthase, cyclooxygenase-2, and vascular endothelial growth factor in LPS-induced RAW264.7 cells were detected by RT-qPCR.

5. The method of using the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids according to claim 1, characterized in that, For the in vivo animal experimental model, female Wistar rats weighing 120±10g were randomly divided into a normal group, a model group, a low-dose lotus leaf alkaloid group, a high-dose lotus leaf alkaloid group, and a positive drug group, with 6 rats in each group. Except for the normal control group, type II collagen-induced arthritis models were established in the other groups of rats. The rats were given a combination containing lotus leaf alkaloid at the set dose. The specific operation steps are as follows: Step 1: During the experiment, the changes in paw volume of rats in each group were measured every 7 days, and arthritis scores were calculated. Step 2: After the last administration, animal samples were collected, and the levels of TNF-α, IL-1β, IL-6, IgG, IgG1, and IgG2a in the serum of rats in each group were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of TNF-α, IL-1β, IL-6, and VEGF in the knee joint tissue of rats were also detected. Step 3: Observe the histopathological changes in the ankle joint of rats in each group by hematoxylin-eosin (H&E) staining and safranin-fast green staining; Step 4: Use X-rays to assess the degree of ankle joint injury and perform imaging scoring, and use Micro-CT scans to scan the rat ankle joint and perform bone parameter analysis.

6. The method of using the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids according to claim 3, characterized in that, When determining the dosage of lotus leaf alkaloids, the patient's genetic testing results are taken into account. For patients with specific gene polymorphisms, the dosage of lotus leaf alkaloids is adjusted based on the correlation studies between genes and drug metabolism, efficacy, and adverse reactions.

7. The method of using the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids according to claim 2, characterized in that, The cellular-level monitoring method employs single-cell sequencing technology to perform single-cell-level gene expression analysis on extracted patient-related cell samples.

8. The method of using the anti-rheumatoid arthritis bone erosion composition containing lotus leaf alkaloids according to claim 4, characterized in that, In the in vitro experimental evaluation process, the evaluation method adopted an extracellular matrix stiffness simulation system to simulate the extracellular matrix stiffness environment of different pathological stages in the joints of patients with rheumatoid arthritis, and to study the effect of lotus leaf alkaloid under different stiffness environments on macrophage inflammatory response.

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