CN111202840A

CN111202840A - Kupffer cell activity inhibitor and application thereof in preparation of drug for treating acute liver failure

Info

Publication number: CN111202840A
Application number: CN201911159458.0A
Authority: CN
Inventors: 胡建军
Original assignee: Shanghai Sixth Peoples Hospital
Current assignee: Shanghai Sixth Peoples Hospital
Priority date: 2019-11-22
Filing date: 2019-11-22
Publication date: 2020-05-29

Abstract

The invention relates to the field of biological medicines, and provides a kupffer cell activity inhibitor which is a recombinant human IL-1 receptor antagonist (rhIL-1 Ra). Experiments show that the rhIL-1Ra can obviously inhibit the activity of large Kupffer cells with high expression of IL-1RI, inhibit the ultrastructure and phagocytic activity of the Kupffer cells, further inhibit the activity of the Kupffer cells, obviously reduce the apoptosis of liver cells and promote the proliferation of the liver cells. In combination with the above conclusions, the recombinant human IL-1 receptor antagonist (rhIL-1Ra) reduces hepatocyte apoptosis and promotes hepatocyte regeneration by inhibiting the activity of Kupffer cells, and provides a strategy for drugs for treating acute liver failure and protecting liver injury.

Description

Kupffer cell activity inhibitor and application thereof in preparation of drug for treating acute liver failure

Technical Field

The invention relates to the field of biological medicines, and particularly relates to a kufu cell activity inhibitor and application thereof in preparation of a medicine for treating acute liver failure.

Background

Acute liver failure is a serious disease with rapid onset and high mortality, and can be caused by conditions such as hepatitis virus, drugs, surgery, etc. (Bernal et al, 2015). In clinical aspect, no medicine for obviously improving the survival rate of patients with acute liver failure exists at present. The only effective method currently recognized for treating acute liver failure is liver transplantation. However, due to the lack of availability of liver sources and the high cost of treatment, this procedure is still not widely applicable (Lee et al, 2015, 2008).

The major members of this family include two active ligands, leukin-1(IL-1 α) and interleukin-1 β (IL-1 β), an antagonist known as IL-1Ra, and two receptors, interleukin-1 receptor type I (IL-1RI) and interleukin-1 receptor type II (IL-1 RII). IL-1Ra and IL-1 have similar affinity to IL-1RI however, unlike IL-1, IL-1Ra is unable to activate receptors and therefore has the effect of inhibiting IL-1 bioactivity IL-1, IL-1Ra, IL-1Ri are highly conserved IL-1 systems, widely involved in a variety of inflammatory responses the ratio of serum IL-1Ra and IL-1 β levels is highly correlated with the outcome of the final inflammatory disease (Dinarelo, 2009, 2011, 2008, NIER).

Kupffer cells are macrophages present in the liver and secrete cytokines such as IL-1 that are closely associated with acute liver failure (cantay et al, 2003; Kolios et al, 2006; Streetz et al, 2000; Zhang et al, 2011). Studies in rats with acute liver failure induced by partial hepatectomy showed that kupffer cells were significantly activated post-operatively, whereas in the control group they appeared morphologically normal (Boermeester et al, 1995). In addition, inhibition of IL-1 activity by IL-1Ra significantly improved pathological changes in liver in acutely liver-exhausted rats, with few activated Kupffer cells found (Streetz et al, 2000). The number of kupffer cells in partially hepatectomy-induced acute liver failure rats was significantly elevated within 72 hours post-surgery, but normal levels were restored only 12 hours post-surgery with the help of platelet-regulated inflammatory cytokines in the kupffer cells (Lopez et al, 2016).

However, at present, whether the activity of the kupffer cell is related to the IL-1 family or not is not reported, and a medicine which can replace liver transplantation operation and can effectively treat acute liver failure is urgently needed to be needed.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a kupffer cell activity inhibitor and application thereof in preparing a medicine for treating acute liver failure. The inhibitor is a recombinant human IL-1 receptor antagonist (rhIL-1Ra), and the experimental result shows that GdCl₃And rhIL-1Ra significantly inhibits phagocytic activity of kupffer cells; under the condition of in vitro co-culture, the kupffer cell obviously inhibits the proliferation of the hepatocyte, so the rhIL-1Ra provides a new strategy for preparing the medicine for treating the acute liver failure.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided an inhibitor of the activity of cells of the Cuvier family, wherein the inhibitor is a recombinant human IL-1 receptor antagonist (rhIL-1 Ra).

Further, the inhibitors inhibit the ultrastructural and phagocytic activity of kupffer cells.

The second aspect of the invention provides the application of the kupffer cell activity inhibitor in preparing a medicament for treating acute liver failure.

Further, the medicine comprises a recombinant human IL-1 receptor antagonist (rhIL-1Ra) and a pharmaceutically acceptable carrier.

Further, the carrier is selected from any one or more of: fillers, binders, solubilizers, disintegrants, and glidants.

Further, the dosage form of the drug is selected from any one of the following: tablet, capsule, pill, granule, suspension, oral liquid and injection.

By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that:

the invention provides a kupffer cell activity inhibitor, namely a recombinant human IL-1 receptor antagonist (rhIL-1 Ra). Because the kupffer cell can obviously inhibit the proliferation of the liver cell, the rhIL-1Ra can inhibit the ultrastructure and phagocytic activity of the kupffer cell, and the rhIL-1Ra further provides a strategy for the medicine for treating the acute liver failure.

Drawings

FIG. 1 is a graph comparing the survival rates of acetaminophen-induced acute liver failure mice treated with rhIL-1Ra and GdCl3 with control mice in accordance with one embodiment of the present invention;

FIG. 2 is a comparison graph of Kupffer cell activity detection of groups of ALF mice in an embodiment of the present invention (wherein, the graph A is a representative graph (x 200) of CD163 immunofluorescence detection, the graph B is a representative graph (x 200) of IL-1RI immunofluorescence detection, the graph C is an overlay effect graph of the graph A and the graph B, the graph D is a comparison graph of the number of large Kupffer cells of each group at 0h, 3h and 6h, the graph E is a histogram of the percentage of large Kupffer cells of each group at 9h, and the graph F is a histogram of the percentage of large Kupffer cells of each group highly expressing IL-1 RI);

FIG. 3 is a graph comparing the ultrastructure (x 5000) of various groups of Kupffer cells and hepatocytes under a transmission electron microscope in one example of the present invention;

FIG. 4 is a phagocytic activity expression graph of the APAP-induced Kupffer cells of ALF mice in each group (wherein, the graphs A-D are representative SPIO-MRI pictures of each treatment group of 3h, 6h, 9h and 0h, respectively; and the graphs E and F are a line graph and a bar graph of the SNR relative variation value of each group, respectively);

FIG. 5 is a graph and a histogram comparing the secretion levels of IL-1 β and IL-1Ra/IL-1 β in each group of early and middle mice in accordance with one embodiment of the present invention (wherein, FIG. A and FIG. B are graphs comparing the secretion levels of IL-1 β in each group with time; FIG. C and FIG. D are graphs comparing the secretion levels of IL-1Ra in each group with time; and FIG. E and FIG. F are graphs comparing the ratio of IL-1Ra/IL-1 β in each group with time);

FIG. 6 is a graph showing a comparison of the effect of each culture condition on the activity of hepatocytes in one example of the present invention (in which FIG. A shows the sorting of Kupffer cells (CD 163) by immunomagnetic bead sorting⁺) A graph of results of (1); FIG. B is a bar graph showing the results of detecting the activity of Kupffer cells and hepatocytes by the CCK8 method, after the groups of cells (Kupffer cell single culture, Kupffer cell and hepatocyte (1:1) layered three-dimensional co-culture, and hepatocyte single culture) were co-cultured with the serum of ALF mice (6 h after APAP molding at 750 mg/kg) diluted at different ratios (1/1, 1/5, 1/10, 1/100, 1/1000) for 48h, respectively.

Detailed Description

The invention provides a kupffer cell activity inhibitor and application thereof in preparing a medicine for treating acute liver failure.

The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.

Antibodies and reagents used in the following examples: acetaminophen, GdCl₃Percoll and collagenase IV were purchased from Sigma of St.Louis, MoAldrich (A7085-100G, G7532-5G, P1644 and C5138), Rabbit polyclonal IL-1RI, sheep polyclonal CD163, donkey anti-rabbit secondary antibodies from san Klaus biochemistry (sc-689, sc-18796, sc-2313), Inc., Santa Crusto, Calif., ELISA kits to detect mIL-1 β and mIL-1Ra from Philid, Wuhan, China (EK0394 and EK0783), Hanks's Balanced Salt Solution (HBSS), fetal bovine serum FBS (FBS) and RPE-1640 (Gibco, Grand Island, NY, USA), superparamagnetic iron oxide (SPIO, Shanghai, China), cell counting kits (CCK8, Dojindo laboratories, Japan, magnetic beads (Miltenyi Biotec, Bergischadbach, Germany), rhIL-1Ra in this laboratory, homology, 0.2 mg, 1997, 1mg of pure Achilles-1 Mg, pure in this laboratory, pure concentration of Achilles-1 Mg, pure II, pure H-1 Mg, pure E-1 Mg, pure E-H-1-H, pure E-H-1 mg, pure E-H-1, pure E-H-1, pure E-H-1, E-H-1, pure E-H-₃It is diluted with sterile physiological saline to a concentration of 3mg/ml before use.

The animals used in the examples below were wild type male C57BL/6 mice, 8-10 weeks old, purchased from Shanghai laboratory animal centers. To avoid the effect of low temperature on liver damage in acute liver failure mice, we kept the ambient temperature at 25 ℃ in animal experiments. The cages were placed randomly, all measures were performed randomly, and the treatment experienced by the observer was unknown for each experimental unit. All animals received humane care and were kept under standard conditions according to protocols approved by the Shanghai university of transportation and legal requirements of China. The Shanghai university of transportation was affiliated with the sixth national hospital animal care and use Committee who prospectively approved this survey. Mice were allowed to acclimate for 1-2 weeks prior to initiation.

Example one

In this example, different treatment groups were constructed, and the relationship between rhIL-1Ra and the survival rate of mice with acute liver failure and the influence of rhIL-1Ra on Kupffer cells were verified at the animal level and the liver histology level. The method comprises the following steps:

1. mortality was observed: all mice (n ═ 39) were injected intraperitoneally with a single 750mg/kg paracetamol injection. After 1 hour, the rhIL-1Ra group (n 10) was injected subcutaneously with 1mg/kg of rhIL-1Ra every 12 hours to 24 hours of acetaminophen injection. GdCl₃Group (n-10) mice were injected subcutaneously with 30mg/kg GdCl 24 hours prior to acetaminophen injection₃. The saline group (n ═ 5) and the acetaminophen group (n ═ 14) were each treated with saline 1 hour after the injection of saline or acetaminophen as controls. The number of deaths after acetaminophen injection was recorded every 2h for each group of mice.

2. Measurement of cytokines in serum: acetaminophen was administered to all mice, and the rhIL-1Ra and GdCl described above were used₃Or normal saline treatment (5 animals per group). at 3, 6 and 9h time points, mouse sera were collected and cytokine levels, including IL-1 β and IL-1Ra, were determined and assayed as directed by the ELISA kit manufacturer.

3. All mice were treated as described above. Superparamagnetic iron oxide-enhanced magnetic resonance (SPIO-MRI) scans of the liver were performed on mice per group (5 mice per group) for 3h, 6h, and 9h, respectively.

4. Study of liver histology: liver tissue specimens were collected uniformly from the same site of the right lobe of the liver for liver histology (specimens fixed in 4% formaldehyde or paraformaldehyde solution at room temperature for 24 hours). Liver samples were cut into small pieces (<0.1 cubic centimeters), fixed in formalin solution, dehydrated, paraffin embedded, and cut to 5 μm thickness. Immunohistochemistry detected the Coffel cell-specific markers IL-1RI and CD 163. Histological analysis was performed by NISI elemental basic research microscope (nikang, shenneca, japan). One slice was taken from the right lobe of each liver, and 7 views (200x) were taken from each slice for calculation.

5. Transmission Electron Microscope (TEM)

Under a transmission electron microscope, liver specimens of each group of mice are quickly cut into small pieces (1 mm)³) Immediately fixed with 4% glutaraldehyde for 24h, then placed with 1% osmic acid for 1-2h, acetone dehydrated and embedded in Epson 812. Preparation of ultrathin sections, in a transmission electron microscope (JEM 1230, JEOL) at 5000 XmagnificationLtd, Tokyo, Japan) to observe the ultrastructure of kupffer cells.

6. Superparamagnetic iron oxide enhanced magnetic resonance scan examination of liver

SPIO-MRI is often used to evaluate the activity of Cuvier cells due to their specific phagocytosis of SPIO (Asanuma et al, 2010; Matos et al, 2015; Tanimoto et al, 2002). The specific operation steps are as follows: each mouse was anesthetized by intraperitoneal injection of 10% chloral hydrate (4 ml/kg). Then, a clinical 3.0T MRI scanner and a four-channel animal coil (Achieva smith-2.1; Philips medical System (Netherlands)) were used. The contrast agent groups were scanned after the start of the injection of superparamagnetic iron oxide (SPIO). These are tail vein injections at a concentration of 30. mu. mol/iron. T1 weighted images (T1W) MRI parameters are: TR/TE 500/23, saline a 8, slice thickness 0.9cm, FOV 50 × 50mm, and stroma 224 × 200. The signal-to-noise ratio (SNR) was calculated by dividing the liver signal intensity by the background noise (water). And calculating the change of the relative signal to noise ratio as (SNRPOST-SNRPRE)/SNRPRE x 100%, wherein SNRPOST is the signal to noise ratio after comparison, and SNRPRE is the signal to noise ratio before comparison.

As can be seen from FIG. 1, rhIL-1Ra and GdCl₃The survival rate of the mice with acute liver failure is obviously improved.

As can be seen from fig. 2, in the previous study (Hu et al, 2010), the most severe liver lesions in the various stages of acute liver failure were found to include hepatocyte eosinophilic degeneration, apoptosis and necrosis, mainly localized in the lobular III region of the liver. Similarly, it was found in this example that large cells of kupffer were also distributed mainly in region III in the three groups at various stages of acute liver failure, although they were distributed mainly in region I in the saline group (fig. 2A, 2C). Suggesting that the activity of the large kupffer cell is closely related to the apoptosis of the liver cell.

In all groups, large Coffel cells highly expressed IL-1RI, and other non-parenchymal cells and hepatocytes only expressed IL-1RI at low levels (FIG. 2B). Whether GdCl₃The group was also rhIL-1Ra, and the number of large cells per stage was significantly reduced compared to the acetaminophen group (FIG. 2D, 2E). Furthermore, the proportion of large Coffet cells highly expressing IL-1RI was significantly higher in all three acute liver failure groups at all stages than in the normal saline groupBut at 3h, 6h and 9h GdCl₃Group (average p)>0.05) and rhIL-1Ra group (s.p.<0.01) the percentage of large Kupffer cells that highly expressed IL-1RI was significantly reduced compared to the APAP group (FIG. 2F). This also further indicates that the large kupffer cell highly expressing IL-1RI is a key target cell for rhIL-1Ra to inhibit the apoptosis of liver cells of mice with acute liver failure.

As can be seen from fig. 3, 0 h: the Kupffer cells of normal mice are morphologically irregular, have a pronounced cytoplasmic processes but are essentially free in the antral cavity, with more primary lysosomes in the cytoplasm. A large number of mitochondria can be seen in the cytoplasm of the hepatocyte, the mitochondrial cristae is short, sparse and clear, and lipid droplets with different sizes and a rough endoplasmic reticulum which are rich and follow the mitochondria can be seen, and more ribosome attachments can be seen on the rough endoplasmic reticulum. 3 h: kupffer cell surface microvilli increase and intracytoplasmic lysosomes increase in the model group. The cytoplasmic matrix of the liver cells is loose, and organelles are scattered; mitochondrial swelling, with blurry cristae; the rough endoplasmic reticulum dilates and has a partial degranulation. GdCl₃The group is similar to the rhIL-1Ra group in lesion, and Kupffer cells have hyperplasia activity expression similar to the model group but are relatively light; the hepatocyte mitochondria in the two groups have light swelling, the cristae is still visible, and the ribosome particles on the rough endoplasmic reticulum are still more visible. 6 h: kupffer cells in the model group are obviously hypertrophied, surface microvilli and intracytoplasmic lysosomes are obviously increased, and more secondary lysosomes are visible. The swelling of the liver cell mitochondria is more obvious, and the ridges are more fuzzy; there were fewer ribosomal particles on the rough endoplasmic reticulum. GdCl₃The Kupffer cells of groups and rhIL-1Ra also seen significant secondary lysosomes but less than in the model group; the swelling of liver cell mitochondria and the ribosome degranulation on the rough endoplasmic reticulum of the two groups are relatively light. 9 h: kupffer cells of the model group are more hypertrophic and occupy hepatic sinuses, a large amount of surface microvilli are visible and reach endothelial cells, and a large amount of large phagosomes are visible. Organelles in the liver cells are scattered, the mitochondria swelling is obvious, and the ridges are fuzzy; few ribosomal particles are on the rough endoplasmic reticulum. GdCl₃Kupffer cells of the group and the rhIL-1Ra group can be obviously hypertrophied and can see larger phagocytes, but the degree of the Kupffer cells is lighter than that of the model group; the swelling of liver cell mitochondria and the degranulation of ribosome on rough endoplasmic reticulum in the two groupsIs significantly lighter. As described above, rhIL-1Ra and GdCl₃Has inhibitory effect on ultrastructure of kupffer cell.

As can be seen from FIG. 4, 1mg/kg of rhIL-1Ra and 30mg/kg of GdCl₃The APAP-induced phagocytic activity of the Kupffer cell of the ALF mouse is obviously inhibited.

As can be seen from FIG. 5, IL-1 β was at a low level in the early and middle stages of acute liver failure and at a high level in the later stage in the acetaminophen group, IL-1 β was always at a low level in the rhIL-1Ra group, and was significantly lower than that in the acetaminophen group GdCl in the late stage of acute liver failure₃The IL-1 β levels in the group were significantly higher in the early stages of acute liver failure than in the rhIL-1Ra group, and were only mildly higher in the mid-late stages (FIGS. 5A, 5B). in all stages of acute liver failure, the IL-1Ra levels in the acetaminophen group were very close to those in the normal saline group₃Group IL-1Ra levels were all higher than those of acetaminophen (FIGS. 5C, 5D).

The IL-1Ra/IL-1 β ratio was kept low for the acetaminophen group, which was not significantly different at the early and middle stages but was lower at the late stage than for the normal saline group₃In the group, only in the late stage, the ratio was significantly higher than in the acetaminophen group (fig. 5E, 5F).

The above data indicate that high levels of endogenous IL-1Ra in the early and middle stages, low levels of IL-1 β in the late stage, and a high IL-1Ra/IL-1 β ratio are associated with rhIL-1Ra functioning to reduce hepatocyte apoptosis.

Example two

In this example, the relationship between kupffer cells and hepatocytes, which were cultured in serum of mice with acute liver failure, was investigated by co-culturing kupffer cells and hepatocytes.

1. Isolation of kupffer cells from hepatocytes: the cells were obtained from wild type male C57BL/6 mice by collagenase digestion and differential centrifugation, and usedThe slightly modified percoll method described previously (Abe et, 2004). Briefly, the liver was perfused with 100ml Ca via the portal vein²⁺And Mg²⁺The HBSS-free perfusion solution of (1) was at a flow rate of 5ml/min at 37 ℃. Followed by perfusion with intact HBSS containing 0.05% collagenase at 37 ℃ for 10 min. The liver was then excised, minced with HBSS/collagenase IV solution, and continuously stirred at 37 deg.C for 20 min. The liver slurry was filtered through a 100 mesh sieve and washed with HBSS. The filtrate was centrifuged at 1000g for 5min, and PBS (pH7.4) was resuspended to a final volume of 10ml, and centrifuged at 500g for 5 min. Collecting the supernatant containing mainly non-parenchymal cells to separate the cells of the Coffeta. The microspheres mainly containing liver cells are revived in RPMI 1640 culture medium, and centrifuged for 5min at 500 g. Cells were counted and diluted to a final concentration of 5X 10 in 5% (v/v) fetal bovine serum in RPMI 1640 medium⁵Per ml, 5% CO at 37 ℃₂And (5) culturing. The supernatant used to isolate the cells of the sink was centrifuged at 500g with 25% and 50% intermittent gradient for 20 min. The cell-enriched fraction of the pool was collected from the boundary layer between the two gradients and washed with RPMI-1640. The sink cell fragments were further purified by antibody CD163 magnetic bead isolation. The cells were cultured at 5X 10⁵The seed was inoculated in 6-well plates at a density of one ml, 37 ℃ and 5% CO ₂15% fetal bovine serum was cultured in RPMI-1640. The survival rate of the separated liver cells reaches more than 91 percent and the survival rate of the kupffer cells reaches more than 95 percent through the detection of a placental blue method.

2. Cell culture: to analyze the interaction between hepatocytes and kupffer cells, we performed a 2-compartment co-culture system. Liver cell (5X 10)⁵) Inoculating to the lower chamber, and placing cells of Kufu (5X 10)⁵) In the upper chamber, the cells were cultured for 24 hours in a recovery culture at 37 ℃ and 5% carbon dioxide atmosphere. The medium of the cell culture was replaced with serum containing 1, 5, 10, 100 and 1000 fold dilutions (induced by 750mg/kg acetaminophen) of acute liver failure mice and incubation was continued for 48 hours. The proliferation activity of hepatocytes was evaluated using CCK8 kit. In addition, hepatocytes (5X 10)⁵) Hekupffer cell (5X 10)⁵) Also cultured in 6-well plates alone and with sera (of acetaminophen-induced acute liver failure mice at 750 mg/kg) at 1, 5, 10, 100 and 1000 fold dilutions for 48 hours, respectively.

3. Statistical analysis: data are expressed as mean ± standard deviation and were statistically analyzed using GraphPad Prism 5 (graphpadsofware, San Diego, CA, USA). The statistical significance of the differences was determined by student's t-test between the two groups and analysis of variance of three or more groups. Survival rates were compared by the Kaplan-Meier method. P values less than 0.05 are significant.

As can be seen from FIG. 6, the ratio of cells in the sink was significantly increased after the inoculation (FIG. 6A). Compared with the culture medium treatment, the acute liver failure mouse serum diluted by different times obviously increases the proliferation of the kupffer cells. However, proliferation of hepatocytes in the co-culture system was significantly inhibited by serum compared to hepatocytes cultured alone (fig. 6B). The above results indicate that activated kupffer cells have a significant inhibitory effect on hepatocyte proliferation.

As described above, rhIL-1Ra inhibits activation of Kupffer cells by binding to IL-1RI of Kupffer cells, thereby inhibiting some pathways of hepatocyte apoptosis, ultimately leading to decreased hepatocyte apoptosis. Based on the mechanism that rhIL-1Ra inhibits the activation of kupffer cells, rhIL-1Ra can provide a strategy for the drugs for treating acute liver failure and liver injury.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. An inhibitor of kupffer cellular activity, wherein said inhibitor is a recombinant human IL-1 receptor antagonist.

2. The inhibitor of the activity of kupffer cells according to claim 1, wherein the inhibitor inhibits the ultrastructural and phagocytic activities of kupffer cells.

3. Use of the kupffer cell activity inhibitor according to any one of claims 1-2 in the preparation of a medicament for the treatment of acute liver failure.

4. Use of an inhibitor of kupffer cell activity according to claim 3, in the manufacture of a medicament for the treatment of acute liver failure, wherein the medicament comprises a recombinant human IL-1 receptor antagonist and a pharmaceutically acceptable carrier.

5. Use of an inhibitor of kupffer cellular activity according to claim 3 in the manufacture of a medicament for the treatment of acute liver failure, wherein the carrier is selected from any one or more of: fillers, binders, solubilizers, disintegrants, and glidants.

6. Use of an inhibitor of the activity of cells of Coffet according to claim 3 in the manufacture of a medicament for the treatment of acute liver failure, wherein the medicament is in a dosage form selected from any one of: tablet, capsule, pill, granule, suspension, oral liquid and injection.