CN116115603A - Application of penicillium purpurogenum lactone C in preparing anti-inflammatory or tissue injury healing promoting medicine - Google Patents

Abstract

The invention discloses an application of producing penicillium C in preparing anti-inflammatory or tissue injury healing promoting drugs, and an application of producing penicillium C in preparing anti-inflammatory or tissue injury healing promoting drugs, wherein the producing penicillium C is shown in a formula I:

in vitro experiments prove that the penicillactone C and the liposome preparation thereof have the function of obviously inhibiting inflammatory factors on mouse macrophages induced by LPS. Meanwhile, the penicillium purpurogenum lactone C and the engineering liposome microneedle preparation remarkably promote the healing of skin wounds of mice in vivo. The obtained penicillium lactone C has anti-inflammatory and tissue injury healing promoting effects. The transcriptome sequencing result shows that the compound can be displayedInhibiting the activity of TLR4 and MyD88 and further inhibiting the expression of downstream NF- κB signaling pathway related proteins.

Description

Application of Violet-producing penicillin C in the preparation of anti-inflammatory or tissue injury-healing medicaments

technical field

The invention belongs to the technical field of medicines, and in particular relates to the application of penicillin C in the preparation of anti-inflammatory or tissue injury-healing medicaments.

Background technique

As a plastic and pluripotent cell population, macrophages exhibit unique phenotypes and functions under the influence of different microenvironments in vivo and in vitro. Studies have shown that macrophages have a series of continuous functional states, and M1 and M2 macrophages are the two extremes of this continuous state (Xu XY et al., Stem Cells Transl Med, 2019, 8, 392-403) . M1 type is classically activated macrophage (classically activated macrophage), M2 type is alternatively activated macrophage (Orecchioni M et al., Front Immunol, 2019, 10, 1084; Shapouri-Moghaddam A et al., J Cell Physiol, 2018, 233, 6425-6440). M1 macrophages secrete pro-inflammatory cytokines and chemokines, and present antigens professionally, participate in the positive immune response, and perform the function of immune surveillance; M2 macrophages only have weak antigen presentation ability, It also down-regulates the immune response by secreting inhibitory cytokines such as IL-10 and TGF, and plays an important role in the later stage of tissue damage repair (Yan J et al., Trends Cell Biol, 2020, 30, 979-989). Under in vitro culture conditions, M1 macrophages are induced by IFN-γ and lipopolysaccharides (LPS) and M2 macrophages are induced by Th2 cytokines (such as IL-4, IL-13, etc.), Possessing similar phenotypes and functions to polarized macrophages in vivo, has become an important means to study macrophage heterogeneity. In different physiological and disease states of the body, macrophages show different types, and identifying macrophage types through phenotypic analysis has become the main method for studying the functional diversity of macrophages (Kadomoto S et al., Int J Mol Sci, 2021 , 23, 144; Elkin M. Adv Exp Med Biol, 2020, 1221, 445-460; KlocM et al., J Tissue Eng Regen Med, 2019, 13, 99-109).

In the past ten years, the polarization disorder of macrophages has a great influence on wound healing, and the elucidation of the mechanisms and targets of macrophages regulating wound healing will help to develop new therapeutic drugs to solve clinical bottlenecks such as inflammation and difficult healing of tissue damage. Studies have shown that M1 macrophage markers are significantly up-regulated in the inflammatory microenvironment, and excessive M1 macrophages increase the expression of pro-inflammatory cytokines and impair the migration of keratinocytes, which is one of the important reasons for inflammatory wound healing disorders (Huang et al. SM et al., J Dermatol Sci, 2019, 96, 159-167). A large number of studies have proved that M1 macrophages and their key activation receptor TLR4 are highly expressed in early healing wound tissue, and play a key role in regulating macrophage-mediated chronic inflammation in wounds (Davis FM et al., J Immunol, 2020, 204, 2503-2513). How to target and regulate the polarization function of local macrophages to promote wound healing is a current clinical research hotspot, and it is also one of the problems to be solved urgently.

Penicillium purpurogenum lactone C is a novel 6/4/5/5 spirocyclic bergamotane-type sesquilide isolated from the rice solid fermentation product of a traditional Chinese medicine endophytic fungus Penicillium purpurogenum Terpene lactone compounds. The authorized Chinese patent ZL201811246712.6 discloses a sesquiterpene compound, a fungal secondary metabolite extract containing it and its use. The penicillin-producing penicillin C shown in formula I of the present invention is the Compound 3. Preliminary pharmacological activity studies found that the compound has a strong function of inhibiting pancreatic lipase activity. There is no literature report about other biological functions of this compound.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide the application of the penicillin-producing penicillin C in the preparation of anti-inflammatory or tissue injury-healing medicaments.

The second object of the present invention is to provide the application of the preparation containing violacinogen lactone C represented by formula I as the only active ingredient in the preparation of anti-inflammatory or tissue injury-healing medicaments.

Technical scheme of the present invention is summarized as follows:

Application of penicillin lactone C in the preparation of anti-inflammation or tissue injury promotion medicament, said penicillin lactone C in purple is shown in formula I:

Application of the preparation with the purpuration-producing penicillin C shown in formula I as the only active ingredient in the preparation of anti-inflammatory or tissue injury-healing medicaments.

The above-mentioned preparation is preferably a liposome preparation or an engineered liposome microneedle preparation.

Advantages of the present invention:

In vitro experiments have proved that penicillin-producing penicillin C and its liposome preparation can significantly inhibit the inflammatory factors TNF-α, IL-6 and IL-1β on LPS-induced mouse macrophages.

At the same time, penicillin C and engineered liposome microneedle preparations significantly promoted skin wound healing in mice in vivo. It is proved that penicillin-producing penicillin C has anti-inflammation and promotes the healing of tissue injury. The results of transcriptome sequencing showed that the compound could significantly inhibit the activities of TLR4 and MyD88, and further inhibit the expression of downstream NF-κB signaling pathway-related proteins to play an anti-inflammatory effect.

The penicillin lactone C-producing liposome preparation and the engineered liposome microneedle preparation represented by the formula I of the present invention can be used as an anti-inflammatory dosage form for treating inflammatory tissue damage.

Description of drawings

Figure 1 is the effect of compound I on inhibiting the inflammatory phenotype activation of LPS-induced mouse macrophage RAW 264.7 and the expression of inflammatory factor TNF-α in vitro (*: p<0.05, **: p<0.01, ns: p>0.05);

Figure 2 shows the results of transcriptome sequencing: (A) compound I significantly inhibited the expression of a series of inflammatory factors and chemokines in macrophages induced by LPS; (B) GSEA analysis showed that compound I significantly down-regulated the Toll-like receptor signaling pathway and Inflammation-related signaling pathways;

Fig. 3 is the effect that compound I and the engineered liposome microneedle preparation prepared by compound I promote diabetic wound healing in vivo;

Figure 4 shows the results of Western blot showing that Compound I and Compound I liposome preparations can significantly inhibit LPS-induced activation of NF-κB signaling pathway in mouse macrophage RAW264.7 at a concentration of 1 μM.

Detailed ways

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Example 1

The penicillin lactone C produced by formula I, referred to as compound I, its preparation method refers to a sesquiterpene compound disclosed in the authorized Chinese patent ZL201811246712.6, a fungal secondary metabolite extract containing it and its purposes, the penicillin lactone C shown in the formula I of the present invention is the compound 3 in the patent document.

Pharmacological experiment

Experimental example 1: Determination of compound I on the activation of LPS-induced mouse macrophage RAW264.7 and the release of inflammatory factors

Instruments: constant temperature incubator, microplate reader, PCR instrument

Reagents: TNF-αELSA Kit, iNOS PCR Kit, Immunohistochemical Staining Kit (Proteintedh)

Experimental steps:

Take the mouse macrophage RAW264.7 in a good growth state and in the logarithmic growth phase (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, and the Cell Resource Center), and the concentration is 5×10 ⁵ cell/mL The cells were seeded in 6-well plates and cultured for 24 hours at 37°C in a 5% CO ₂ incubator. After 24h, compound I was added to make the final concentrations of 10nM, 1μM and 1mM respectively. After 1h, LPS (final concentration 1μg/mL, solvent PBS) was added, and the blank group and LPS group were set up at 37°C, 5% CO ₂ cultured for 36h. After 36 hours, the RNA of each group was extracted, and the effect of compound I on the expression level of the inflammatory phenotype marker iNOS of mouse macrophage RAW264.7 induced by LPS was detected according to the standard operation procedure of the iNOS PCR kit manual. At the same time, the culture supernatants of each group were collected, and the effect of compound I on the level of inflammatory factor TNF-α released by LPS-induced mouse macrophages RAW264.7 was detected according to the standard operating procedures of the TNF-α ELSA kit instructions. As shown in 1, compound I can significantly inhibit LPS-induced activation of mouse macrophage RAW264.7 (Fig. 1A, B, D, E) and the release of inflammatory factor TNF-α at a concentration of 1 μM (Fig. 1C).

Experimental example 2: The key pathway of compound I regulating macrophages is the TLR4-NFκB pathway

Instrument: Workstation of Guangzhou Jidio Biotechnology Co., Ltd.

Reagent: Miltenyi Biotec Macrophage Isolation Kit

Experimental steps:

The primary peritoneal macrophages of mice in good growth state were sorted and identified according to the instructions of the macrophage sorting kit using 6-week-old C57BL/6 mice, and the mice were purchased from Speiford (Beijing) Biotechnology Co., Ltd.), cells with a concentration of 5×10 ⁵ cell/mL were inoculated in a 6-well plate, and cultured in a 5% CO ₂ incubator at 37° C. for 24 hours. After 24h, compound I was added to make the final concentration 1μM. After 1h, LPS (final concentration 1μg/mL, solvent PBS) was added, and the blank group and LPS group were set at the same time, and incubated at 37°C, 5% CO ₂ for 36h. After 36 hours, the medium supernatant was discarded, and the cells were washed with pre-cooled PBS buffer solution. The cells were collected, snap-frozen in liquid nitrogen, and sent to the workstation of Guangzhou Gideo Biotechnology Co., Ltd. for transcriptome sequencing analysis. The results are shown in Figure 2, compound I significantly inhibited the expression of a series of inflammatory factors and chemokines in macrophages induced by LPS (Figure 2A). GSEA analysis showed that the compounds significantly downregulated the Toll-like receptor pathway, inflammation-related pathways and TNF-related pathways (Fig. 2B). The classic pathway of LPS-induced macrophages is the TLR4-NFκB pathway, and the results suggest that compound I may play a key regulatory role in the activation of this pathway.

Experimental example 3

Compound I and its engineered liposome microneedle preparation target regulation of macrophage function to promote diabetic wound healing Instrument: Workstation

Reagents: ultraviolet (UV) photoinitiator, GelMA solution, hyaluronic acid solution

Experimental steps:

Compound I engineered liposome microneedle preparation is prepared by compound I liposome preparation further equipped with GelMA microneedle preparation, compound I liposome preparation and compound I engineered liposome microneedle preparation preparation method is as follows:

The preparation method of Compound I liposome preparation: RAW264.7 cells were subjected to centrifugation and hypotonic treatment to separate the cell membrane, then 10 μM Compound I solution (solvent PBS) was separated and extracted with the macrophage cell membrane solution (10 mg/mL concentration, 1 mL of the system, the solvent PBS) was hydrated for 30 minutes, during which the loading between Compound I and the vesicles was facilitated by repeated freeze-thaw cycles. Finally, the Compound I liposome formulation with a particle size of 100 nm was extruded using an Avanti micro extruder and a polycarbonate porous membrane. The transmission electron micrograph of compound I liposome preparation is shown in Figure 3A, the particle size is shown in Figure 3B, and the infrared spectrum is shown in Figure 3C.

The preparation method of Compound I engineered liposome microneedle preparation: 100 μ L of the GelMA solution (purchased from EFL) was poured onto a negative microneedle mold of polydimethylsiloxane (PDMS). Air bubbles were removed by vacuum and centrifuged at 3000 rpm for 20 minutes. The material was then exposed to 350mW/cm2 UV light (405nm) for 60s to facilitate curing. Finally, 5% (w/v) hyaluronic acid solution (EFL, Suzhou, Jiangsu, China) was added to prepare the microneedle base, incubated at 30-35°C for more than 12 hours, and then demolded. The photomicrograph of compound I engineered liposome microneedle preparation is shown in Figure 3D, the scanning electron micrograph is shown in Figure 3E, and the drug release curve is shown in Figure 3F.

A mouse model of type 2 diabetes was established by feeding on a high-fat diet for 6 months. Fasting blood glucose monitoring and glucose tolerance testing were used to confirm the success of the model, and it was further found that the skin wound healing of diabetic mice was delayed. Immediately after wound modeling, mice in the diabetes+compound I engineered liposome microneedle group were treated with Compound I engineered liposome microneedle preparation locally (non-treated mice in the healthy group and diabetes group were given Compound I engineered mice only). Adjuvant treatment of liposome microneedle preparation, diabetes + compound I injection group given the same dose of compound I local injection), found that compound I engineered liposome microneedle preparation successfully achieved the effect of promoting diabetic wound healing, and its curative effect was superior In excipient treatment and local direct injection of Compound I (Fig. 3G, Fig. 3H).

Experimental Example 4: Compound I and compound I liposome preparations have effects on iNOS, p-p65 NF-κB, p65 NF-κB, p-IκBα, IκBα, p-IκK and Effect of IκK protein expression level assay

Instruments: electrophoresis apparatus, electrotransfer apparatus

Reagents: iNOS, p-p65 NF-κB, p65 NF-κB, p-IκBα, IκBα, p-IκK and IκK antibodies and Western blot routine reagents

Experimental steps:

Take the mouse macrophage RAW264.7 in a good growth state and in the logarithmic growth phase, inoculate the cells at a concentration of 5×10 ⁵ cell/mL in a 6-well plate, and incubate at 37°C, 5% CO ₂ Cultivate for 24h. After 24 hours, add 1 μM compound I to the compound I group, add 1 μM compound I liposome preparation to the compound I liposome group, add 1 μM empty liposome to the empty liposome group, and add 1 μM empty liposome to the above groups after 1 hour. LPS (final concentration 1 μg/mL) (solvent PBS) was added to the LPS group, and a blank control group was set at the same time, and cultured at 37° C., 5% CO ₂ for 36 hours. After 36 hours, discard the medium supernatant, wash the cells with pre-cooled PBS buffer, collect the cells, add cell lysate, protease and phosphatase inhibitors, extract protein, and use BCA protein concentration assay kit for protein quantification. According to the routine Western blot experimental procedures, 7.5% polyacrylamide gel electrophoresis (SDS-PAGE) was used to perform electrophoresis and membrane transfer with a protein loading amount of 30 μg per well. Block the membrane with 5% BSA or 5% skimmed milk powder, then add primary antibodies (iNOS, p-p65 NF-κB, p65 NF-κB, p-IκBα, IκBα, p-IκK and IκK), at 4°C After incubation for 12 hours, wash with TBST buffer for 3 times, then add peroxidase (HRP)-labeled secondary antibody and incubate at room temperature for 1 hour, and finally use a chemiluminescence kit for luminescence detection.

As shown in Figure 3, Compound I (Figure 4A) and Compound I liposome formulation (Figure 4B) can significantly inhibit LPS-induced iNOS, p-p65 NF- The expression of κB, p-IκBα and p-IκK indicated that the compound could inhibit the activation of NF-κB signaling pathway, thereby exerting its anti-inflammatory effect.

Compound I of the present invention, according to conventional technical means, and pharmaceutically acceptable carriers and/or excipients, is made into preparations suitable for oral administration, injection or local application, etc., for example, liposome microneedle preparations, Or add pharmaceutically acceptable carriers and/or excipients to form tablets, capsules, powders, syrups, injections, etc. according to conventional techniques.

Compositions containing compound I of the present invention have pharmacological activity.

The descriptions of the above embodiments are only used to help understand the method and central idea of the present invention. It should be pointed out that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall under the protection of the claims of the present invention.

Claims (3)

1. The application of producing penicillin C in the preparation of anti-inflammation or promoting tissue injury healing medicine, said producing penicillin C as shown in formula (I):

2. The application of the preparation containing the violacin lactone C represented by the formula (I) as the only active ingredient in the preparation of anti-inflammatory or tissue injury-healing medicaments. 3. The application according to claim 2, characterized in that the preparation is a liposome preparation or an engineered liposome microneedle preparation.

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