Preparation method of nardostachys jatamansi self-assembled particles for regulating lung cell aging based on p53 or p16 pathway

Abstract

The application relates to the technical field of medicine and pharmacy, and discloses a preparation method of a nardostachy self-assembled particle for regulating lung cell aging based on a p53 or p16 channel, which comprises the following steps: S1, soaking nardostachy decoction pieces in water, and then heating and decocting to obtain a decoction; S2, centrifuging the decoction obtained in the step S1 for the first time, and collecting supernatant; S3, performing freezing treatment on the supernatant obtained in the step S2, and then naturally thawing to obtain a thawed liquid; S4, centrifuging the thawed liquid obtained in the step S3 for the second time, collecting the precipitate, and washing the precipitate with purified water to obtain the nardostachy self-assembled particle. Through a specific freeze-thaw self-assembling process, nanoscale spherical particles are successfully prepared from the nardostachy decoction pieces, the particles have an average particle size of 50-500 nm, have good dispersibility and stability, the problems that traditional Chinese medicine extracts are complex in components and difficult to be standardized are solved, and efficient enrichment and controllable production of active components are realized.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for preparing Notopterygium incisum self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence. Background Technology

[0002] Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are common critical illnesses in clinical practice, with a mortality rate as high as 30%–40%. Its core pathological mechanisms include damage to the alveolar epithelium and pulmonary microvascular endothelium, disruption of the alveolar-capillary barrier, non-cardiac pulmonary edema, excessive recruitment of inflammatory cells, and progressive hypoxemia. Current clinical treatment primarily focuses on supportive care, such as lung-protective ventilation, restrictive fluid management, and prone positioning ventilation, with a lack of specific drug therapies.

[0003] Current drug treatments have the following main problems: Firstly, while broad-spectrum anti-inflammatory drugs such as glucocorticoids can suppress the inflammatory response, they carry the risks of immunosuppression and secondary infection, and the evidence of their efficacy is inconsistent, making it impossible to effectively intervene in the multi-faceted network pathology of ALI.

[0004] Secondly, targeted antagonists against single inflammatory factors (such as TNF-α, IL-1β, etc.) have repeatedly failed in clinical trials. This is because ALI is not driven by a single pathway, but is a network pathological process involving multiple intertwined links such as inflammation amplification, barrier destruction, matrix remodeling and repair barriers. Inhibiting a single node cannot reverse the overall pathological process.

[0005] Thirdly, recent studies have found that during ALI, alveolar epithelial cells, endothelial cells, and immune cells undergo p53 / p16-mediated senescence-like activation under inflammation and oxidative stress, releasing senescence-associated secretory phenotype (SASP) factors, further amplifying inflammation, exacerbating barrier damage, and delaying repair. However, existing senescence intervention drugs mostly promote apoptosis of senescent cells, which has problems such as hematologic toxicity and insufficient cell selectivity, and there is no mature protocol for ALI yet. In addition, ALI is an acute disease, and large-scale removal of senescent cells may simultaneously damage cells needed for repair. What is needed is to inhibit the activation of senescence-like programs rather than to remove cells, and current technologies lack such gentle regulatory means.

[0006] Fourthly, Notopterygium root is a traditional Chinese medicine. Its dried rhizome and root have the effects of dispelling wind and cold, removing dampness and relieving pain. Modern pharmacological research focuses on its analgesic, anti-inflammatory, antipyretic, and antioxidant properties, and it is mainly used for rheumatoid arthritis, osteoarthritis, etc. Currently, there is no technical solution for using self-assembled particles derived from Notopterygium root to treat acute lung injury, nor are there any traditional Chinese medicine-derived drugs that achieve lung protection by inhibiting the p53 / p16-SASP program.

[0007] In view of this, we propose a method for preparing Notopterygium incisum self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing self-assembled particles of Notopterygium incisum based on the regulation of lung cell senescence by the p53 or p16 pathway. This method uses a specific freeze-thaw self-assembly process to extract self-assembled particles with a nanoscale spherical particle morphology from Notopterygium incisum slices. These particles can achieve multi-link, network-like lung protection by inhibiting cell senescence activation and SASP secretion mediated by the p53 / p16 pathway.

[0009] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway regulating lung cell senescence, the method comprising: S1. Soak the sliced ​​Qianghuo herbs in water, then heat and decoct to obtain the decoction; S2. Centrifuge the decoction obtained in step S1 for the first time and collect the supernatant. S3. Freeze the supernatant obtained in step S2, and then thaw it naturally to obtain the thawed liquid; S4. Centrifuge the thawed solution obtained in step S3 for the second time, collect the precipitate, and wash the precipitate with purified water to obtain the self-assembled particles of Notopterygium incisum.

[0010] Preferably, in step S1, the weight ratio of the Qianghuo medicinal slices to water is 1:5 to 1:15, the soaking time is 0.5 to 2 hours, and the decoction time is 0.5 to 2 hours.

[0011] Preferably, the simmering in step S1 is a gentle boiling simmering at a temperature of 95–105°C.

[0012] Preferably, the centrifugal force for the first centrifugation in step S2 is 8,000 to 12,000 × g, and the centrifugation time is 10 to 20 minutes.

[0013] Preferably, the freezing temperature in step S3 is -25℃ to -15℃, and the freezing time is 12 to 24 hours; the natural thawing temperature is 18 to 30℃, and the thawing time is 1 to 4 hours.

[0014] Preferably, the centrifugal force of the second centrifugation in step S4 is 8,000 to 12,000 × g, and the centrifugation time is 10 to 20 minutes; the number of washing cycles is 2 to 4.

[0015] Preferably, after step S4, the method further includes drying the obtained Qianghuo self-assembled particles, wherein the drying is freeze-drying or vacuum drying, and the moisture content of the dried particles is ≤5%.

[0016] Preferably, the average particle size of the self-assembled Notopterygium incisum particles obtained in step S4 is 50-500 nm, and the Zeta potential is -30 mV to -10 mV.

[0017] Preferably, the polysaccharide content of the self-assembled granules of Notopterygium incisum obtained in step S4 is ≥40%, the total phenol content is ≥5%, and it contains at least two active ingredients from chlorogenic acid, ferulic acid, and isoimperatorin.

[0018] Preferably, the self-assembled particles of Notopterygium incisum obtained in step S4 are used to prepare drugs that inhibit cell senescence activation and senescence-related secretory phenotypes mediated by the p53 / p16 pathway.

[0019] By employing the above technical solution, this invention provides a method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence. It possesses at least the following beneficial effects: This invention successfully prepared nanoscale spherical particles from Notopterygium root slices through a specific freeze-thaw self-assembly process. The average particle size of these particles is 50-500 nm, and they exhibit good dispersibility and stability. This invention solves the problem of complex composition and difficulty in standardization of traditional Chinese medicine extracts, and achieves efficient enrichment and controllable production of active ingredients.

[0020] By inhibiting p53 / p16 pathway-mediated cellular senescence activation and SASP secretion, this invention achieves comprehensive protection against multiple pathways in acute lung injury. It addresses issues such as severely impaired lung function, abnormally elevated airway resistance, and pulmonary edema in the model group, significantly reducing pathological changes in lung tissue, including alveolar septal thickening, inflammatory cell infiltration, and alveolar hemorrhage. These effects are not achieved by broad-spectrum anti-inflammatory drugs such as glucocorticoids because this invention does not rely on immunosuppression but rather exerts its effects through network regulation, thus resolving the issues of immunosuppression and secondary infection risks associated with existing broad-spectrum anti-inflammatory drugs.

[0021] Transcriptome sequencing revealed that nanoscale spherical particles reversely regulate 536 differentially expressed genes, significantly enriched in multiple pathways such as cytokine-cytokine receptor interactions, cell adhesion molecules, chemotaxis, immune responses, and inflammatory responses, achieving a holistic remodeling of the immune-inflammatory network. This differs from single cytokine antagonists, which can only inhibit a single target, and overcomes the limitation of single-target drugs in reversing the network pathology of ALI.

[0022] This invention addresses the problems of hematologic toxicity, insufficient cell selectivity, and potential damage to cells requiring repair during acute disease processes by inhibiting p53 / p16-related cellular senescence activation and SASP inflammatory secretion programs, rather than by eliminating senescent cells. This is different from the apoptosis-promoting mechanism of senolytic drugs. Attached Figure Description

[0023] The accompanying drawings, which are provided to further illustrate the invention, constitute a part of this application: Figure 1 The diagram (A) and scanning electron microscope image (B) of the test substance acquisition process of the method for preparing Notopterygium incisum self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence of the present invention are shown. Figure 2 The present invention describes the improvement effect of QH-FSAPs on lung tissue pathology, W / D ratio, Cdyn and RI by the preparation method of Qianghuo self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence. Figure 3 Transcriptome analysis of the preparation method of Notopterygium incisum self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence in this invention is shown, including 536 differentially expressed genes with reverse regulation and KEGG / GO enrichment results. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1: Obtaining the test compound QH-FSAPs Take 50g of Notopterygium root slices, soak in double-distilled water at room temperature for 1 hour, then simmer over low heat for 1 hour. Centrifuge the decoction at 10,000×g for 15 minutes, discard the dregs and keep the supernatant. Freeze the supernatant at -20℃ for 18 hours (or overnight), then thaw naturally at room temperature. Centrifuge the thawed solution at 10,000×g for 15 minutes, collect the precipitate, and resuspend and wash three times with double-distilled water. The resulting precipitate is QH-FSAPs, which should be resuspended in sterile physiological saline before administration. Scanning electron microscopy shows that it has a nanoscale spherical particle morphology (see Appendix). Figure 1 ; Example 2: Verification of its use in treating acute lung injury Modeling: Male C57BL / 6J mice aged 8–10 weeks were anesthetized with 2% sodium pentobarbital (50 mg / kg), fixed in a supine position with the neck slightly extended; the glottis was exposed under cold light illumination, and LPS 2.5 mg / kg (dissolved in 50 μL) was instilled into the trachea through a 20G cannula; the sham-operated group was given an equal volume of PBS.

[0026] Administration: Two hours after modeling, QH-FSAPs were prepared with sterile saline and administered orally at a dose of 4 g / kg and 250 μL / animal.

[0027] Evaluation (24 hours after modeling): Lung function tests (respiratory rate 110 breaths / min, tidal volume 10 mL / kg, inspiratory-to-expiratory ratio 1.5:1), Cdyn and RI were measured; W / D ratio was measured in the left lower lobe; HE staining and lung injury scoring (edema, interstitial inflammatory infiltration, hemorrhage, atelectasis, hyaline membrane formation) were performed in the right upper lobe.

[0028] Results: QH-FSAPs significantly improved all of the above indicators; Example 3: Optional technical solutions for effective dosage Three dosage groups were established: 2 g / kg (low), 4 g / kg (medium), and 8 g / kg (high). Results showed that 2 g / kg provided weaker protection; 4 g / kg produced stable protection; and 8 g / kg showed no additional benefit superior to 4 g / kg. Therefore, 4 g / kg is the preferred dosage, and the dosage of this invention can be 2–8 g / kg, preferably 3–6 g / kg. Example 4: Optional technical solutions for timing and course of drug administration Therapeutic administration (validated): Administer the medication 2 hours after injury and evaluate the patient 24 hours later. This timing simulates the actual clinical scenario of a patient presenting with lung injury, thus having stronger clinical relevance.

[0029] Example 5: Verification of the Mechanism of Action 5.1 Transcriptome analysis: Bulk RNA-seq was performed on lung tissues from three groups. PCA showed good separation between groups. Differential expression analysis and intersection analysis revealed 536 reverse regulatory genes (217 of which were upregulated by LPS and downregulated by QH-FSAPs, and 319 were the reverse). KEGG / GO enrichment is shown in Figure 3. 5.2 Detection of aging biomarkers: Western blot was used to detect p53 and p16 proteins in lung tissue (primary antibody 1:1000, HRP-labeled secondary antibody 1:5000, β-actin as internal control). LPS significantly upregulated p53 (P=0.0047) and p16 (P<0.0001); QH-FSAPs significantly downregulated p53 (P=0.0243) and p16 (P<0.0001). 5.3 SASP program analysis: GSEA was performed based on the REACTOME gene set, and the SASP gene set was significantly enriched; a heatmap of SASP characteristic genes was drawn based on the SAUL_SEN_MAYO gene set, showing LPS upregulation and QH-FSAPs callback; 5.4 Hub Gene Analysis: PPI network analysis showed that Mmp9 is located in the core interaction module, suggesting it is a candidate hub gene. [Mmp9 requires further verification through protein expression and enzyme activity.] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0030] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway regulating lung cell senescence, characterized in that, The method includes: S1. Soak the sliced ​​Qianghuo herbs in water, then heat and decoct to obtain the decoction; S2. Centrifuge the decoction obtained in step S1 for the first time and collect the supernatant. S3. Freeze the supernatant obtained in step S2, and then thaw it naturally to obtain the thawed liquid; S4. Centrifuge the thawed solution obtained in step S3 for the second time, collect the precipitate, and wash the precipitate with purified water to obtain the self-assembled particles of Notopterygium incisum.

2. The method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence according to claim 1, characterized in that, In step S1, the weight ratio of the Qianghuo medicinal slices to water is 1:5 to 1:15, the soaking time is 0.5 to 2 hours, and the decoction time is 0.5 to 2 hours.

3. The method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence according to claim 1, characterized in that, The simmering process described in step S1 is a gentle, slow simmering process at a temperature of 95–105°C.

4. The method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence according to claim 1, characterized in that, The centrifugal force for the first centrifugation in step S2 is 8,000 to 12,000 × g, and the centrifugation time is 10 to 20 minutes.

5. The method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence according to claim 1, characterized in that, The freezing temperature in step S3 is -25℃ to -15℃, and the freezing time is 12 to 24 hours; the natural thawing temperature is 18 to 30℃, and the thawing time is 1 to 4 hours.

6. The method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence according to claim 1, characterized in that, In step S4, the centrifugal force for the second centrifugation is 8,000–12,000 × g, and the centrifugation time is 10–20 minutes; the washing is performed 2–4 times.

7. The method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence according to claim 1, characterized in that, Step S4 is followed by a step of drying the obtained Qianghuo self-assembled particles, wherein the drying is freeze-drying or vacuum drying, and the moisture content of the dried particles is ≤5%.

8. The method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence according to claim 1, characterized in that, The average particle size of the self-assembled Notopterygium incisum particles obtained in step S4 is 50-500 nm, and the Zeta potential is -30 mV to -10 mV.

9. The method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence according to claim 1, characterized in that, The self-assembled granules of Notopterygium incisum obtained in step S4 have a polysaccharide content of ≥40%, a total phenol content of ≥5%, and contain at least two active ingredients from chlorogenic acid, ferulic acid, and isoimperatorin.

10. The method for preparing *Notopterygium incisum* self-assembled particles based on the p53 or p16 pathway to regulate lung cell senescence according to claim 1, characterized in that, The self-assembled particles of Notopterygium incisum obtained in step S4 are used to prepare drugs that inhibit cell senescence activation and senescence-related secretory phenotypes mediated by the p53 / p16 pathway.

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