Application of an immunomodulator for inhibiting over-activation of astrocytes in neuropathic pain

Abstract

The present invention discloses the application of an immunomodulator for inhibiting the overactivation of astrocytes in neuropathic pain, which comprises Lactobacillus plantarum CGMCC 1.12935 and Streptococcus thermophilus CGMCC 1.1481. A natural immunomodulator containing matrine is prepared by fermenting Sophora flavescens, which can be applied to neuropathic pain, can effectively inhibit the overactivation of astrocytes, and thus achieve the effect of reducing pain.

Description

Technical Field

[0001] The present invention relates to the application of an immunomodulator for inhibiting the over-activation of astrocytes in neuropathic pain, and belongs to the technical field of microbiology. Background Art

[0002] Neuropathic pain is a pain directly caused by damage or disease involving the somatosensory system. This disease is common and is often considered a chronic pain disease caused by spinal cord injury or primary damage to the peripheral and central nerves. It seriously affects the quality of life of patients and also affects their mental health and social functions. However, this disease is often misdiagnosed clinically and ordinary analgesics, such as non-steroidal anti-inflammatory drugs, are given, resulting in little efficacy and delayed treatment. Common diseases that cause neuropathic pain include: diabetic polyneuropathy, postherpetic neuralgia, postoperative neuralgia, etc. Modern research has found that the activation of spinal cord astrocytes induced by neuroinflammation may be the direct cause of persistent neuropathic pain. And the expression of glial fibrillary acidic protein (GFAP) in the central nervous system is closely related to the activation of astrocytes. Some studies have shown that the up-regulation of GFAP expression in the nervous system is directly proportional to the activation of astrocytes.

[0003] Inflammation is a way for the human body to protect itself, used to eliminate harmful stimuli, including damaged cells and pathogens. Existing research has shown that inflammation plays an important role in neuropathic pain and chronic neurodegenerative diseases. Many natural compound drug sources have characteristics such as low price, diverse biological activities, and few adverse reactions. They can regulate immunity, resist excessive damage to the human body caused by inflammation, and reduce many side effects brought about by treating diseases. Probiotics are a type of microorganism that is basically non-toxic and has no side effects on the human body. The regulatory effect of probiotics on the intestinal flora, the protective effect on the intestinal barrier, and the impact on intestinal immune function have been widely reported. Astragalus polysaccharide, lentinan, and ginsenoside obtained by probiotic fermentation have been applied in clinical immunotherapy as immunomodulators. Sophora flavescens, as a common Chinese medicinal material native to China, is recorded in medical books such as "Shennong Ben Cao Jing" and "Compendium of Materia Medica". Sophora flavescens is cold in nature and bitter in taste, and has the effects of clearing heat and detoxifying, removing heat and drying dampness, killing insects and expelling worms, etc. Modern research has found that the main active ingredients of Sophora flavescens are alkaloids and flavonoid compounds, including oxymatrine, oxysophocarpine, matrine, trifoliol, santalin, kushenone, etc., which have good analgesic, anti-tumor, and antibacterial activities.

[0004] The present invention aims to provide a natural immunomodulator prepared by fermenting Sophora flavescens, which can be applied to neuropathic pain and can effectively inhibit the over-activation of astrocytes, thereby achieving the effect of reducing pain. Summary of the Invention

[0005] The object of the present invention is to provide a natural immunomodulator prepared by fermenting Sophora flavescens, which can be applied to neuropathic pain and can effectively inhibit the over-activation of astrocytes, thereby achieving the effect of reducing pain.

[0006] All the bacteria used in this patent are commercially available.

[0007] The Lactobacillus plantarum, this strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC 1.12935.

[0008] The Lactobacillus plantarum, this strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC 1.296.

[0009] The Streptococcus thermophilus, this strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC 1.1481.

[0010] The Streptococcus thermophilus, this strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC 1.2718.

[0011] Matrine is mainly used for the treatment of hepatitis and tumors clinically. Matrine has pharmacological effects such as inhibiting inflammation, scavenging free radicals, protecting the liver, immune regulation, and neuroprotection, and may have good clinical application value in improving metabolism and immune regulation of the body. The applicant's team found that using Lactobacillus plantarum CGMCC 1.12935 alone can improve the fermentation quality of Sophora flavescens to a certain extent and enhance its immune regulation effect, while using Streptococcus thermophilus CGMCC 1.1481 alone to ferment Sophora flavescens has no obvious effect.

[0012] The R & D team further studied that if Lactobacillus plantarum CGMCC 1.12935 and Streptococcus thermophilus CGMCC 1.1481 are used for co-fermenting Sophora flavescens, it can have a relatively excellent immunosuppressive effect of inhibiting the over-activation of astrocytes and can better relieve the neuropathic pain of rats.

[0013] The technical problems to be solved by the present invention can be achieved through the following technical solutions.

[0014] Preparation of a natural immune regulator:

[0015] Preparation of bacterial strain seed liquid (the inoculation amounts described in the present invention are all v / v):

[0016] Lactobacillus plantarum CGMCC 1.12935: The preserved Lactobacillus plantarum CGMCC 1.12935 strain was thawed, streaked on MRS solid medium and cultured at 37 °C for 24 hours. The Lactobacillus plantarum strain with obvious colony characteristics was picked and inoculated into MRS liquid medium, and cultured at 200 r / min and 37 °C for 24 hours. The bacterial liquid concentration was adjusted to 1×10 7 CFU / ml Lactobacillus plantarum seed liquid.

[0017] Streptococcus thermophilus CGMCC 1.1481: The preserved Streptococcus thermophilus CGMCC 1.1481 strain was thawed, streaked on MRS solid medium and cultured at 37 °C for 24 hours. The Streptococcus thermophilus strain with obvious colony characteristics was picked and inoculated into MRS liquid medium, and cultured at 200 r / min and 37 °C for 24 hours. The bacterial liquid concentration was adjusted to 1×10 7 CFU / ml Streptococcus thermophilus seed liquid.

[0018] Preparation of Sophora flavescens fermentation extract:

[0019] (1) An appropriate amount of Sophora flavescens medicinal materials were dried, ground and passed through a 40-mesh sieve. 1 part by weight of the ground Sophora flavescens was taken, 5 parts by weight of distilled water was added, 1% of Lactobacillus plantarum seed liquid was inoculated, 1% of Streptococcus thermophilus seed liquid was inoculated, and fermented at 37 °C for 24 hours.

[0020] (2) Then 10 parts by weight of 75% ethanol was added, and reflux extraction was carried out at 75 °C for 1 time. Another 10 parts by weight of 75% ethanol was added, and reflux extraction was carried out at 75 °C for 1 time. The filtrates were combined, concentrated, passed through a 0.22-μm filter membrane, and rotary evaporated to a thick state, and then freeze-dried under vacuum at -80 °C to obtain a yellowish-white powder. It was stored in a sealed manner at 4 °C.

[0021] Advantages of the present invention:

[0022] (1) Both Streptococcus thermophilus and Lactobacillus plantarum are food-grade bacteria with high safety, and the safety is guaranteed.

[0023] (2) When the two bacteria are co-fermented with Sophora flavescens, they can inhibit the over-activation of astrocytes, reduce the inflammatory response, and better relieve the neuropathic pain of rats. Specific implementation mode

[0024] Embodiments of the present invention will be described in detail below. These embodiments are only used to explain the present invention and should not be construed as limiting the present invention.

[0025] Example 1

[0026] Preparation of a natural immune regulator:

[0027] Preparation of strain seed liquid (all inoculation amounts described in the present invention are v / v):

[0028] Lactobacillus plantarum CGMCC 1.12935: The preserved Lactobacillus plantarum CGMCC 1.12935 strain was thawed, streaked on MRS solid medium and cultured at 37 °C for 24 hours. A Lactobacillus plantarum strain with obvious colony characteristics was picked and inoculated into MRS liquid medium, and cultured at 200 r / min and 37 °C for 24 hours. The bacterial liquid concentration was adjusted to 1×10 7 CFU / ml Lactobacillus plantarum seed liquid.

[0029] Streptococcus thermophilus CGMCC 1.1481: The preserved Streptococcus thermophilus CGMCC 1.1481 strain was thawed, streaked on MRS solid medium and cultured at 37 °C for 24 hours. A Streptococcus thermophilus strain with obvious colony characteristics was picked and inoculated into MRS liquid medium, and cultured at 200 r / min and 37 °C for 24 hours. The bacterial liquid concentration was adjusted to 1×10 7 CFU / ml Streptococcus thermophilus seed liquid.

[0030] Preparation of Sophora flavescens fermentation extract:

[0031] (1) An appropriate amount of Sophora flavescens medicinal materials were dried, ground and passed through a 40-mesh sieve. 1 part by weight of the ground Sophora flavescens was taken, 5 parts by weight of distilled water was added, 1% of Lactobacillus plantarum seed liquid was inoculated, 1% of Streptococcus thermophilus seed liquid was inoculated, and fermented at 37 °C for 24 hours.

[0032] (2) Then 10 parts by weight of 75% ethanol was added and refluxed at 75 °C for 1 time. Another 10 parts by weight of 75% ethanol was added and refluxed at 75 °C for 1 time. The filtrates were combined, concentrated, passed through a 0.22-μm filter membrane, rotary evaporated to a thick state and then freeze-dried under vacuum at -80 °C to obtain a yellowish-white powder. It was stored in a sealed manner at 4 °C.

[0033] Example 2

[0034] Preparation of a natural immune regulator, which only contains Lactobacillus plantarum CGMCC 1.12935, and the others are the same as in Example 1.

[0035] Preparation of a natural immune regulator:

[0036] Preparation of strain seed liquid (all inoculation amounts described in the present invention are v / v):

[0037] Lactobacillus plantarum CGMCC 1.12935: The preserved Lactobacillus plantarum CGMCC 1.12935 strain was thawed, streaked on MRS solid medium and cultured at 37°C for 24 hours. The Lactobacillus plantarum strain with obvious colony characteristics was picked and inoculated into MRS liquid medium, and cultured at 200 r / min and 37°C for 24 hours. The concentration of the bacterial liquid was adjusted to 1×10 7 CFU / ml Lactobacillus plantarum seed liquid.

[0038] Preparation of Sophora flavescens fermentation extract:

[0039] (1) An appropriate amount of Sophora flavescens medicinal materials were dried, ground and passed through a 40-mesh sieve. 1 part by weight of the ground Sophora flavescens was taken, 5 parts by weight of distilled water was added, and 2% of the Lactobacillus plantarum seed liquid was inoculated, and fermented at 37°C for 24 hours.

[0040] (2) Then 10 parts by weight of 75% ethanol was added and refluxed at 75°C for 1 time. Another 10 parts by weight of 75% ethanol was added and refluxed at 75°C for 1 time. The filtrates were combined, concentrated, passed through a 0.22-μm filter membrane, rotary evaporated to a thick state and then freeze-dried under vacuum at -80°C to obtain a yellowish-white powder. It was stored in a sealed manner at 4°C.

[0041] Example 3

[0042] Preparation of a natural immune regulator, which only contains Streptococcus thermophilus CGMCC 1.1481, and the others are the same as in Example 1.

[0043] Preparation of a natural immune regulator:

[0044] Preparation of bacterial strain seed liquid (the inoculation amounts described in the present invention are all v / v):

[0045] Streptococcus thermophilus CGMCC 1.1481: The preserved Streptococcus thermophilus CGMCC 1.1481 strain was thawed, streaked on MRS solid medium and cultured at 37°C for 24 hours. The Streptococcus thermophilus strain with obvious colony characteristics was picked and inoculated into MRS liquid medium, and cultured at 200 r / min and 37°C for 24 hours. The concentration of the bacterial liquid was adjusted to 1×10 7 CFU / ml Streptococcus thermophilus seed liquid.

[0046] Preparation of Sophora flavescens fermentation extract:

[0047] (1) An appropriate amount of Sophora flavescens medicinal materials were dried, ground and passed through a 40-mesh sieve. 1 part by weight of the ground Sophora flavescens was taken, 5 parts by weight of distilled water was added, and 2% of the Streptococcus thermophilus seed liquid was inoculated, and fermented at 37°C for 24 hours.

[0048] (2) Add 10 parts by weight of 75% ethanol and reflux for extraction once at 75°C. Add another 10 parts by weight of 75% ethanol and reflux for extraction once at 75°C. Combine the filtrates, concentrate the filtrates, filter through a 0.22 μm filter membrane, rotary evaporate to a thick consistency, and then freeze-dry under vacuum at -80°C to obtain a yellowish-white powder. Store it in a sealed container at 4°C.

[0049] Example 4

[0050] Preparation of a natural immune regulator: Replace Lactobacillus plantarum CGMCC 1.12935 with Lactobacillus plantarum CGMCC 1.296, and the others are the same as in Example 1.

[0051] Preparation of a natural immune regulator:

[0052] Preparation of the strain seed liquid (the inoculation amounts described in the present invention are all v / v):

[0053] Lactobacillus plantarum CGMCC 1.296: Thaw the preserved Lactobacillus plantarum CGMCC 1.296 strain, streak it on an MRS solid medium and culture it for 24 hours at 37°C. Pick a Lactobacillus plantarum strain with obvious colony characteristics and inoculate it into an MRS liquid medium, culture it at 200 r / min and 37°C for 24 hours, and adjust the bacterial liquid concentration to 1×10 7 CFU / ml of Lactobacillus plantarum seed liquid.

[0054] Streptococcus thermophilus CGMCC 1.1481: Thaw the preserved Streptococcus thermophilus CGMCC 1.1481 strain, streak it on an MRS solid medium and culture it for 24 hours at 37°C. Pick a Streptococcus thermophilus strain with obvious colony characteristics and inoculate it into an MRS liquid medium, culture it at 200 r / min and 37°C for 24 hours, and adjust the bacterial liquid concentration to 1×10 7 CFU / ml of Streptococcus thermophilus seed liquid.

[0055] Preparation of the Sophora flavescens fermentation extract:

[0056] (1) Appropriate amount of Sophora flavescens medicinal materials are dried and ground through a 40-mesh sieve. Take 1 part by weight of the ground Sophora flavescens, add 5 parts by weight of distilled water, inoculate 1% of the Lactobacillus plantarum seed liquid, inoculate 1% of the Streptococcus thermophilus seed liquid, and ferment at 37°C for 24 hours.

[0057] (2) Add 10 parts by weight of 75% ethanol and reflux for extraction once at 75°C. Add another 10 parts by weight of 75% ethanol and reflux for extraction once at 75°C. Combine the filtrates, concentrate the filtrates, filter through a 0.22 μm filter membrane, rotary evaporate to a thick consistency, and then freeze-dry under vacuum at -80°C to obtain a yellowish-white powder. Store it in a sealed container at 4°C.

[0058] Example 5

[0059] Preparation of a natural immune regulator: Replace Streptococcus thermophilus CGMCC 1.1481 with Streptococcus thermophilus CGMCC 1.2718, and the others are the same as in Example 1.

[0060] Preparation of a natural immune regulator:

[0061] Preparation of bacterial strain seed liquid (the inoculation amounts described in the present invention are all v / v):

[0062] Lactobacillus plantarum CGMCC 1.12935: Thaw the preserved Lactobacillus plantarum CGMCC 1.12935 strain, streak it on an MRS solid medium and culture it. Culture it for 24 hours at 37°C, pick a Lactobacillus plantarum strain with obvious colony characteristics and inoculate it into an MRS liquid medium, culture it at 200 r / min and 37°C for 24 hours, and adjust the bacterial liquid concentration to 1×10 7 CFU / ml of Lactobacillus plantarum seed liquid.

[0063] Streptococcus thermophilus CGMCC 1.2718: Thaw the preserved Streptococcus thermophilus CGMCC 1.2718 strain, streak it on an MRS solid medium and culture it. Culture it for 24 hours at 37°C, pick a Streptococcus thermophilus strain with obvious colony characteristics and inoculate it into an MRS liquid medium, culture it at 200 r / min and 37°C for 24 hours, and adjust the bacterial liquid concentration to 1×10 7 CFU / ml of Streptococcus thermophilus seed liquid.

[0064] Preparation of Sophora flavescens fermentation extract:

[0065] (1) Appropriate amount of Sophora flavescens medicinal materials are dried, ground and passed through a 40-mesh sieve. Take 1 part by weight of the ground Sophora flavescens, add 5 parts by weight of distilled water, inoculate 1% of Lactobacillus plantarum seed liquid and 1% of Streptococcus thermophilus seed liquid, and ferment at 37°C for 24 hours.

[0066] (2) Then add 10 parts by weight of 75% ethanol and reflux extract at 75°C for 1 time. Add 10 parts by weight of 75% ethanol again and reflux extract at 75°C for 1 time. Combine the filtrates, concentrate the filtrates and pass them through a 0.22-μm filter membrane, rotary evaporate to a thick state and then freeze-dry under vacuum at -80°C to obtain a yellowish-white powder. Store it in a sealed manner at 4°C.

[0067] Example 6

[0068] Preparation of a natural immune regulator:

[0069] Preparation of Sophora flavescens fermentation extract:

[0070] (1) Appropriate amount of Sophora flavescens medicinal materials are dried, ground and passed through a 40-mesh sieve. Take 1 part by weight of the ground Sophora flavescens, add 5 parts by weight of distilled water, and incubate at 37°C for 24 hours.

[0071] (2) Then add 10 parts by weight of 75% ethanol and reflux extract at 75 °C for 1 time. Add another 10 parts by weight of 75% ethanol and reflux extract at 75 °C for 1 time. Combine the filtrates, concentrate the filtrates, filter through a 0.22 μm filter membrane, rotary evaporate until thick, and then freeze-dry under vacuum at -80 °C to obtain a yellowish-white powder. Store it airtight at 4 °C.

[0072] Example 7

[0073] Preparation of a neuropathic pain model: Use the chronic constriction injury method to induce neuropathic pain in rats. Select male SD rats with a body weight of 200 - 250 g. Rats are routinely anesthetized and disinfected. Incise the skin of the left hind limb and then incise under the femoral muscle to expose the sciatic nerve and its three branches. Ligate the sciatic nerve and its branches, and suture the muscle layer and skin of the surgical area. If the rats show phenomena such as limping and the hind limb hanging in the air, it indicates that the rat model is successfully established. In the sham operation group, only the sciatic nerve is isolated and exposed without ligation. Set the sham operation group as the negative group and the model group as the positive group. The sham operation group and the model group are given intraperitoneal injection of normal saline. The experimental group is the group of rats with the model treated with drugs. Inject the Sophora flavescens fermentation extract obtained from the groups of Examples 1 - 6 into the modeled rats at a dose of 100 mg / kg by intraperitoneal injection, once a day for 10 consecutive days. There are a total of 8 groups, with 5 rats in each group. On the 11th day, the mechanical withdrawal threshold measurement and the thermal withdrawal latency measurement are carried out.

[0074] The mechanical withdrawal threshold (MWT) test detects the response of rats to mechanical stimuli: The animals are placed in a chamber, and the left hind paw of each rat is stimulated with von Frey filaments. The stimulation time is about 3 - 5 s, with an interval of 30 s. After the up and down force stimulation, direct a 0.6 g von Frey force to the plantar surface of the hind paw. A positive response is defined as encountering the stimulus and the hind paw immediately retracting. If the foot retreats, reduce the induced force; on the contrary, when a negative response occurs, increase the induced force. Repeat the above process until the minimum force that can cause the rat's paw to retract is determined. Take the von Frey force that can cause the paws of 50% of the rats to retract as the withdrawal threshold. Each animal undergoes the MWT test 10 times. The cut-off value of the von Frey force is set at 15 g, and the results are shown in Table 1.

[0075] Table 1 Results of MWT measurement

[0076] Group MWT (g) Negative control group (sham operation group) 13.16±1.81 Positive control group (model group) 2.03±0.42 Example 1 group <![CDATA[11.45±1.51 1，2，3 > Example 2 group <![CDATA[7.05±0.78 1，2，3 > Example 3 group <![CDATA[5.24±0.65 1，2 > Example 4 group <![CDATA[5.35±0.62 1，2 > Example 5 group <![CDATA[7.15±0.98 1，2，3 > Example 6 group 5.07±0.59

[0077] Note: t-test, 1: P < 0.05 (compared with the negative control group); 2: P < 0.05 (compared with the positive control group); 3: P < 0.05 (compared with the group of Example 6)

[0078] The thermal withdrawal latency (TWL) test was used to detect the sensitivity of the paw to thermal stimulation. Rats were placed on the surface of a 3-mm thick glass plate, covered with a plexiglass chamber, and a BME-410C fully automatic plantar analgesia meter was used. After the left hind paw of the rat was subjected to thermal stimulation, the duration of withdrawing the paw from the heat source was considered the TWL. For each test, five thermal stimulations were given every 5 min, and the average TWL was calculated. The set cut-off value was 30 s, and the results are shown in Table 2.

[0079] Table 2 Results of TWL determination

[0080] Group TWL (s) Negative control group (sham operation group) 22.32±2.42 Positive control group (model group) 5.35±0.68 Example 1 group <![CDATA[19.65±2.12 1，2，3 > Example 2 group <![CDATA[12.54±1.76 1，2，3 > Example 3 group <![CDATA[9.87±1.08 1，2 > Example 4 group <![CDATA[9.99±1.24 1，2 > Example 5 group <![CDATA[13.45±1.84 1，2，3 > Example 6 group 9.42±0.99

[0081] Note: t-test, 1: P < 0.05 (compared with the negative control group); 2: P < 0.05 (compared with the positive control group); 3: P < 0.05 (compared with the group of Example 6)

[0082] The results showed that compared with the sham operation group, the MWT and TWL of the rats in the model group were significantly shortened, and the MWT and TWL of the rats treated with Sophora flavescens extract were significantly increased. Compared with the group of Example 6 (single ethanol extract of Sophora flavescens), the groups of Example 2 and 5 (containing Lactobacillus plantarum CGMCC 1.12935) could significantly increase the mechanical withdrawal threshold and thermal withdrawal latency, while the groups of Example 3 (containing Streptococcus thermophilus CGMCC 1.1481) and Example 4 (containing other types of Lactobacillus and Streptococcus thermophilus CGMCC 1.1481) had no obvious effect, indicating that the fermentation product of Lactobacillus plantarum CGMCC 1.12935 has its uniqueness and can further improve the immune activity of the natural fermentation product of Sophora flavescens, while other types of Lactobacillus or single use of Streptococcus thermophilus cannot achieve such an effect; and the extract obtained by fermenting Sophora flavescens with both Lactobacillus plantarum CGMCC 1.12935 and Streptococcus thermophilus CGMCC 1.1481 at the same time had the strongest immune activity, with significantly increased MWT and TWL, approaching those of the rats in the sham operation group. It is considered that only the special fermentation of Streptococcus thermophilus CGMCC 1.1481 can produce a synergistic effect with Lactobacillus plantarum CGMCC 1.12935, thus changing the fermentation process of Sophora flavescens by Lactobacillus plantarum CGMCC 1.12935 and improving the fermentation quality of the fermentation broth of Sophora flavescens.

[0083] Example 8

[0084] After the rat tests were completed, the rats were induced to anesthesia, perfused with normal saline through the heart, and the spinal cord region between L4-L6 segments was taken for subsequent experiments.

[0085] GFAP Observation: Frozen sections were fixed. The sections were incubated overnight at 4°C with the primary anti-GFAP antibody (dilution: 1:200), then the secondary antibody was added, and incubated at 37°C for 30 min. After color development, counterstaining and mounting, the sections were observed under a microscope. The protein expression level of GFAP was analyzed using Image J software, and the results are shown in Table 3.

[0086] Table 3 Relative protein expression of GFAP

[0087] Group Relative protein expression Negative control group (sham operation group) 0.42±0.05 Positive control group (model group) 1.35±0.12 Example 1 group <![CDATA[0.60±0.06 1，2，3 > Example 2 group <![CDATA[0.90±0.10 1，2，3 > Example 3 group <![CDATA[0.97±0.11 1，2 <!-- 6 -->]]> Example 4 group <![CDATA[0.95±0.08 1，2 > Example 5 group <![CDATA[0.88±0.10 1，2，3 > Example 6 group 1.04±0.10

[0088] Note: t-test, 1: P < 0.05 (compared with the negative control group); 2: P < 0.05 (compared with the positive control group); 3: P < 0.05 (compared with the group of Example 6)

[0089] Determination of inflammatory factors: ELISA was used to detect the levels of inflammatory factors. The spinal cord segments of another 6 rats in each group were frozen quickly in liquid nitrogen, ground, and made into homogenates. After centrifugation, the supernatant was taken, and IL-1β and TNF-α were detected using an ELISA kit. The optical density value at 490 nm was measured using an enzyme-labeled instrument, and the average expression levels of IL-1β and TNF-α were analyzed. The results are shown in Table 4.

[0090] Table 4 Determination of inflammatory factor levels (pg / mL)

[0091] Group IL-1β TNF-α Negative control group (sham operation group) 9.02±0.82 37.24±6.96 Positive control group (model group) 50.53±5.35 219.46±19.77 Example 1 group <![CDATA[11.26±1.50 1，2，3 > <![CDATA[53.21±5.26 1，2，3 > Example 2 group <![CDATA[28.65±3.29 1，2，3 > <![CDATA[113.26±13.99 1，2，3 > Example 3 group <![CDATA[34.26±3.66 1，2 > <![CDATA[151.18±12.89 1，2 > Example 4 group <![CDATA[38.25±4.08 1，2 > <![CDATA[150.26±16.78 1，2 > Example 5 group <![CDATA[27.65±3.11 1，2，3 > <![CDATA[108.26±14.28 1，2，3 > Example 6 group 38.12±3.26 159.26±15.22

[0092] Note: t-test, 1: P < 0.05 (compared with the negative control group); 2: P < 0.05 (compared with the positive control group); 3: P < 0.05 (compared with the group of Example 6)

[0093] Compared with the sham operation group, the relative protein content of GFAP in the spinal cord of rats in the model group was the highest, and the levels of inflammatory factors such as IL-1β and TNF-α were the highest. Compared with the groups in Example 6 (single Sophora flavescens alcohol extract), the levels of inflammatory factors and GFAP in the groups of Example 2 and 5 (containing Lactobacillus plantarum CGMCC1.12935) decreased significantly, while the effects of the groups in Example 3 (containing Streptococcus thermophilus CGMCC 1.1481) and Example 4 (containing other types of lactobacilli and Streptococcus thermophilus CGMCC 1.1481) were not obvious, indicating that the fermentation product of Lactobacillus plantarum CGMCC 1.12935 has its uniqueness and can further improve the immunological activity of the fermentation product of natural Sophora flavescens; while the extract obtained by fermenting Sophora flavescens with both Lactobacillus plantarum CGMCC 1.12935 and Streptococcus thermophilus CGMCC 1.1481 has the strongest ability to reduce the activity of immune factors, and the decrease of GFAP is the largest, approaching that of the rats in the sham operation group. The expression of GFAP protein was significantly up-regulated, and the number of activated astrocytes increased. After adding Sophora flavescens for immunomodulation, the inflammatory factors decreased, and the over-activation of GFAP was inhibited, and the number of activated astrocytes decreased significantly. Considering that Streptococcus thermophilus CGMCC 1.1481 combined with Lactobacillus plantarum CGMCC 1.12935 fermented Sophora flavescens, a synergistic effect may be produced, and more effective small molecule substances may be produced, greatly improving the fermentation quality of the Sophora flavescens fermentation broth.

[0094] In summary, during the research and practice process of the present invention team, the applicant's research team found that using Lactobacillus plantarum CGMCC 1.12935 alone can improve the fermentation quality of Sophora flavescens to a certain extent, while using Streptococcus thermophilus CGMCC1.1481 alone to ferment Sophora flavescens has no obvious effect. However, when the two bacteria are co-fermented, they can have a relatively excellent immunosuppressive effect of inhibiting the over-activation of astrocytes and can better relieve the neuropathic pain of rats.

[0095] The description of terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0096] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. An immunomodulator for inhibiting the over-activation of astrocytes, and its preparation method is as follows: (1) Appropriate amount of Sophora flavescens Ait. herbs are dried and ground through a 40-mesh sieve. Take 1 part by weight of the ground Sophora flavescens Ait., add 5 parts by weight of distilled water, inoculate 1% of the seed liquid of Lactobacillus plantarum CGMCC 1.12935, inoculate 1% of the seed liquid of Streptococcus thermophilus CGMCC 1.1481, and ferment at 37 °C for 24 hours; (2) Then add 10 parts by weight of 75% ethanol, and reflux extract at 75 °C for 1 time; add 10 parts by weight of 75% ethanol again, and reflux extract at 75 °C for 1 time; combine the reflux extracts, filter the reflux extracts through a 0.22-μm filter membrane, rotate and evaporate to a thick state, and then freeze-dry under vacuum at -80 °C to obtain a yellowish-white powder; store it in a sealed manner at 4 °C.

2. The immunomodulator according to claim 1, wherein the preparation method of the seed liquid of Lactobacillus plantarum CGMCC 1.12935 is as follows: The preserved Lactobacillus plantarum CGMCC 1.12935 strain was thawed and streaked on MRS solid medium for cultivation. It was cultured at 37°C for 24 hours. The Lactobacillus plantarum strain with obvious colony characteristics was picked and inoculated into MRS liquid medium, and cultured at 200 r / min and 37°C for 24 hours. The concentration of the bacterial liquid was adjusted to 1×10 7 CFU / ml Lactobacillus plantarum seed liquid.

3. The immunomodulator according to claim 1, wherein the preparation method of the seed liquid of Streptococcus thermophilus CGMCC 1.1481 is as follows: The preserved Streptococcus thermophilus strain CGMCC 1.1481 was thawed, streaked on MRS solid medium and cultured at 37°C for 24 hours. The Streptococcus thermophilus strain with obvious colony characteristics was picked and inoculated into MRS liquid medium, and cultured at 200 r / min and 37°C for 24 hours. The bacterial liquid concentration was adjusted to 1×10 7 CFU / ml of Streptococcus thermophilus seed liquid.

4. The immunomodulator according to any one of claims 1 to 3, characterized in that, The application of the immunomodulator in the preparation of a drug for treating neuropathic pain.