Application of immunomodulator for inhibiting overactivity of astrocytes in neuropathic pain

Abstract

The invention discloses an application of an immunomodulator for inhibiting overactivity of astrocytes in neuropathic pain. The immunomodulator comprises lactobacillus plantarum CGMCC (China General Microbiological Culture Collection Center) 1.12935 and streptococcus thermophilus CGMCC 1.1481. The matrine-containing natural immunomodulator is prepared by fermenting radix sophorae flavescentis, can be applied to neuropathic pain and can effectively inhibit overactivity of astrocytes, so that the effect of relieving pain is achieved.

Description

Technical Field

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

[0002] Neuropathic pain refers to pain directly caused by a certain injury or disease involving the somatic sensory system. This disease is common and is often considered to be a chronic pain disease caused by spinal cord injury or primary damage to the peripheral and central nervous systems. It seriously affects the patient's quality of life and also affects the patient's mental health and social function. Despite this, the disease is often misdiagnosed in clinical practice, 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, and postoperative neuralgia. Modern research has found that neuroinflammation-induced activation of spinal astrocytes may be the direct cause of persistent neuropathic pain. The expression of glial fibrillary acidic protein (GFAP) in the central nervous system is closely related to the activation of astrocytes. Studies have shown that the upregulation of GFAP expression in the nervous system is positively correlated with the activation of astrocytes.

[0003] Inflammation is a way for the human body to protect itself, to remove harmful stimuli, including damaged cells and pathogens. Existing studies have shown that inflammation plays an important role in neuropathic pain and chronic neurodegenerative diseases. Many natural compound sources are low-cost, diverse in biological activity, and have few adverse reactions. They can regulate immunity, resist excessive damage to the human body caused by inflammation, and reduce the many side effects caused by treating diseases. Probiotics are a type of microorganism that has basically no toxic side effects on the human body. The regulatory effect of probiotics on intestinal flora, the protective effect on the intestinal barrier, and the effect on intestinal immune function have been widely reported. Astragalus polysaccharides, Lentinan polysaccharides, and ginsenosides obtained by probiotic fermentation have been used in clinical immunotherapy as immunomodulators. Sophora flavescens, as a common medicinal material native to China, is recorded in medical books such as "Shennong's Herbal Classic" and "Compendium of Materia Medica". Sophora flavescens is cold in nature and bitter in taste. It has the effects of removing heat and detoxification, removing heat and dampness, and killing insects and repelling parasites. Modern research has found that the main active ingredients of Sophora flavescens are alkaloids and flavonoid compounds. Including oxymatrine, oxysophocarpine, matrine, trifoliate purple, santalin, matrine ketone, 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 excessive activation of astrocytes, thereby achieving the effect of alleviating pain. Summary of the invention

[0005] The purpose 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 excessive activation of astrocytes, thereby achieving the effect of alleviating pain.

[0006] The bacteria used in this patent are all commercially available.

[0007] The Lactobacillus plantarum strain is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC 1.12935.

[0008] The Lactobacillus plantarum strain is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC 1.296.

[0009] The Streptococcus thermophilus strain is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC 1.1481.

[0010] The Streptococcus thermophilus strain is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC 1.2718.

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

[0012] The research and development team further studied that if Lactobacillus plantarum CGMCC 1.12935 and Streptococcus thermophilus CGMCC1.1481 are co-fermented with Sophora flavescens, it can have a more excellent immunosuppressive effect of inhibiting the excessive activation of astrocytes and can better alleviate the neuropathic pain in rats.

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

[0014] Preparation of a natural immunomodulator:

[0015] Preparation of bacterial seed solution (the inoculation amount described in the present invention is v / v):

[0016] Lactobacillus plantarum CGMCC 1.12935: Thaw the preserved Lactobacillus plantarum CGMCC 1.12935 strain, streak it on MRS solid medium, culture it at 37°C for 24 hours, pick out the Lactobacillus plantarum strain with obvious colony characteristics and inoculate it into MRS liquid medium, culture it at 200r / min and 37°C for 24 hours, and adjust the bacterial solution concentration to 1*10 7 CFU / ml Lactobacillus plantarum seed solution.

[0017] Streptococcus thermophilus CGMCC 1.1481: Thaw the preserved Streptococcus thermophilus CGMCC 1.1481 strain, streak it on MRS solid medium, culture it at 37℃ for 24 hours, pick out the Streptococcus thermophilus strain with obvious colony characteristics and inoculate it into MRS liquid medium, culture it at 200r / min and 37℃ for 24 hours, and adjust the bacterial solution concentration to 1*10 7 CFU / ml of Streptococcus thermophilus seed solution.

[0018] Preparation of Sophora flavescens fermented extract:

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

[0020] (2) Add 10 parts by weight of 75% ethanol and extract under reflux at 75°C once. Add 10 parts by weight of 75% ethanol again and extract under reflux at 75°C once. Combine the filtrates, concentrate the filtrates and filter through a 0.22um filter membrane. Rotary evaporate until thick and freeze-dry under vacuum at -80°C to obtain a yellow-white powder. Store in a sealed container at 4°C.

[0021] Advantages of the present invention:

[0022] (1) Streptococcus thermophilus and Lactobacillus plantarum are both highly safe food bacteria with guaranteed safety.

[0023] (2) When the two bacteria were co-fermented with Sophora flavescens, they could inhibit the overactivation of astrocytes, reduce the inflammatory response, and effectively alleviate the neuropathic pain in rats. DETAILED DESCRIPTION

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

[0025] Example 1

[0026] Preparation of a natural immunomodulator:

[0027] Preparation of bacterial seed solution (the inoculation amount described in the present invention is v / v):

[0028] Lactobacillus plantarum CGMCC 1.12935: Thaw the preserved Lactobacillus plantarum CGMCC 1.12935 strain, streak it on MRS solid medium, culture it at 37°C for 24 hours, pick out the Lactobacillus plantarum strain with obvious colony characteristics and inoculate it into MRS liquid medium, culture it at 200r / min and 37°C for 24 hours, and adjust the bacterial solution concentration to 1*10 7 CFU / ml Lactobacillus plantarum seed solution.

[0029] Streptococcus thermophilus CGMCC 1.1481: Thaw the preserved Streptococcus thermophilus CGMCC 1.1481 strain, streak it on MRS solid medium, culture it at 37℃ for 24 hours, pick out the Streptococcus thermophilus strain with obvious colony characteristics and inoculate it into MRS liquid medium, culture it at 200r / min and 37℃ for 24 hours, and adjust the bacterial solution concentration to 1*10 7 CFU / ml of Streptococcus thermophilus seed solution.

[0030] Preparation of Sophora flavescens fermented extract:

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

[0032] (2) Add 10 parts by weight of 75% ethanol and extract under reflux at 75°C once. Add 10 parts by weight of 75% ethanol again and extract under reflux at 75°C once. Combine the filtrates, concentrate the filtrates and filter through a 0.22um filter membrane. Rotary evaporate until thick and freeze-dry under vacuum at -80°C to obtain a yellow-white powder. Store in a sealed container at 4°C.

[0033] Example 2

[0034] A preparation method of a natural immunomodulator, containing only Lactobacillus plantarum CGMCC 1.12935, and the other parts are the same as those in Example 1.

[0035] Preparation of a natural immunomodulator:

[0036] Preparation of bacterial seed solution (the inoculation amount described in the present invention is v / v):

[0037] Lactobacillus plantarum CGMCC 1.12935: Thaw the preserved Lactobacillus plantarum CGMCC 1.12935 strain, streak it on MRS solid medium, culture it at 37°C for 24 hours, pick out the Lactobacillus plantarum strain with obvious colony characteristics and inoculate it into MRS liquid medium, culture it at 200r / min and 37°C for 24 hours, and adjust the bacterial solution concentration to 1*10 7 CFU / ml Lactobacillus plantarum seed solution.

[0038] Preparation of Sophora flavescens fermented extract:

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

[0040] (2) Add 10 parts by weight of 75% ethanol and extract under reflux at 75°C once. Add 10 parts by weight of 75% ethanol again and extract under reflux at 75°C once. Combine the filtrates, concentrate the filtrates and filter through a 0.22um filter membrane. Rotary evaporate until thick and freeze-dry under vacuum at -80°C to obtain a yellow-white powder. Store in a sealed container at 4°C.

[0041] Example 3

[0042] A preparation method of a natural immunomodulator, containing only Streptococcus thermophilus CGMCC 1.1481, and the other parts are the same as those in Example 1.

[0043] Preparation of a natural immunomodulator:

[0044] Preparation of bacterial seed solution (the inoculation amount described in the present invention is v / v):

[0045] Streptococcus thermophilus CGMCC 1.1481: Thaw the preserved Streptococcus thermophilus CGMCC 1.1481 strain, streak it on MRS solid medium, culture it at 37℃ for 24 hours, pick out the Streptococcus thermophilus strain with obvious colony characteristics and inoculate it into MRS liquid medium, culture it at 200r / min and 37℃ for 24 hours, and adjust the bacterial solution concentration to 1*10 7 CFU / ml of Streptococcus thermophilus seed solution.

[0046] Preparation of Sophora flavescens fermented extract:

[0047] (1) An appropriate amount of Sophora flavescens is dried and ground to pass through a 40-mesh sieve. 1 weight part of the ground Sophora flavescens is added to 5 weight parts of distilled water, and inoculated with 2% of thermophilic Streptococcus seed solution, and fermented at 37° C. for 24 hours.

[0048] (2) Add 10 parts by weight of 75% ethanol and extract under reflux at 75°C once. Add 10 parts by weight of 75% ethanol again and extract under reflux at 75°C once. Combine the filtrates, concentrate the filtrates and filter through a 0.22um filter membrane. Rotary evaporate until thick and freeze-dry under vacuum at -80°C to obtain a yellow-white powder. Store in a sealed container at 4°C.

[0049] Example 4

[0050] A preparation method of a natural immunomodulator, wherein Lactobacillus plantarum CGMCC 1.12935 is replaced by Lactobacillus plantarum CGMCC 1.296, and the rest is the same as in Example 1.

[0051] Preparation of a natural immunomodulator:

[0052] Preparation of bacterial seed solution (the inoculation amount described in the present invention is v / v):

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

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

[0055] Preparation of Sophora flavescens fermented extract:

[0056] (1) An appropriate amount of Sophora flavescens was dried and ground to pass through a 40-mesh sieve. 1 weight part of the ground Sophora flavescens was taken, 5 weight parts of distilled water was added, and 1% of Lactobacillus plantarum seed liquid and 1% of Streptococcus thermophilus seed liquid were inoculated. The mixture was fermented at 37° C. for 24 hours.

[0057] (2) Add 10 parts by weight of 75% ethanol and extract under reflux at 75°C once. Add 10 parts by weight of 75% ethanol again and extract under reflux at 75°C once. Combine the filtrates, concentrate the filtrates and filter through a 0.22um filter membrane. Rotary evaporate until thick and freeze-dry under vacuum at -80°C to obtain a yellow-white powder. Store in a sealed container at 4°C.

[0058] Example 5

[0059] A preparation method of a natural immunomodulator, wherein the thermophilic Streptococcus CGMCC 1.1481 is replaced by the thermophilic Streptococcus CGMCC 1.2718, and the other steps are the same as those in Example 1.

[0060] Preparation of a natural immunomodulator:

[0061] Preparation of bacterial seed solution (the inoculation amount described in the present invention is v / v):

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

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

[0064] Preparation of Sophora flavescens fermented extract:

[0065] (1) An appropriate amount of Sophora flavescens was dried and ground to pass through a 40-mesh sieve. 1 weight part of the ground Sophora flavescens was taken, 5 weight parts of distilled water was added, and 1% of Lactobacillus plantarum seed liquid and 1% of Streptococcus thermophilus seed liquid were inoculated. The mixture was fermented at 37° C. for 24 hours.

[0066] (2) Add 10 parts by weight of 75% ethanol and extract under reflux at 75°C once. Add 10 parts by weight of 75% ethanol again and extract under reflux at 75°C once. Combine the filtrates, concentrate the filtrates and filter through a 0.22um filter membrane. Rotary evaporate until thick and freeze-dry under vacuum at -80°C to obtain a yellow-white powder. Store in a sealed container at 4°C.

[0067] Example 6

[0068] Preparation of a natural immunomodulator:

[0069] Preparation of Sophora flavescens fermented extract:

[0070] (1) An appropriate amount of Sophora flavescens was dried and ground to pass through a 40-mesh sieve. 1 part by weight of the ground Sophora flavescens was added to 5 parts by weight of distilled water and incubated at 37° C. for 24 hours.

[0071] (2) Add 10 parts by weight of 75% ethanol and extract under reflux at 75°C once. Add 10 parts by weight of 75% ethanol again and extract under reflux at 75°C once. Combine the filtrates, concentrate the filtrates and filter through a 0.22um filter membrane. Rotary evaporate until thick and freeze-dry under vacuum at -80°C to obtain a yellow-white powder. Store in a sealed container at 4°C.

[0072] Example 7

[0073] Preparation of neuropathic pain model, using chronic constriction injury method to induce neuropathic pain in rats. SD male rats with a body weight of 200-250g were selected. Rats were routinely anesthetized and disinfected, the skin of the left hind limb was incised, and the sciatic nerve and its three branches were exposed under the femoral muscle. The sciatic nerve and its branches were ligated, and the muscle layer and skin of the surgical area were sutured. If the rats showed lameness, hind limbs hanging in the air, etc., it indicated that the rat model was successful. The sham operation group only separated and exposed the sciatic nerve without ligation. The sham operation group was set as the negative group and the model group as the positive group. The sham operation group and the model group were given intraperitoneal injection of normal saline. The experimental group was the modeling rat medication group, and the fermented extract of Sophora flavescens obtained in Example 1-6 groups was intraperitoneally injected into the modeling rats at a dose of 100 mg / kg, once / d, for 10 consecutive days. There were a total of 8 groups, each with 5 rats, and mechanical withdrawal threshold and thermal withdrawal latency were measured on the 11th day.

[0074] The mechanical withdrawal threshold (MWT) test examined the rats’ responses to mechanical stimulation: the animals were placed in a small chamber and the left hind paw of each rat was stimulated using a von Frey filament. The stimulation lasted approximately 3-5 seconds with an interval of 30 seconds. After the upper and lower force stimulation, a 0.6 g von Frey force was directed to the plantar surface of the hind foot. A positive response was defined as the immediate withdrawal of the hind paw upon encountering the stimulus. If the foot was withdrawn, the induced force was reduced; conversely, when a negative response occurred, the induced force was increased. The above process was repeated until the minimum force that could cause the rats’ paws to withdraw was determined. The von Frey force that could cause 50% of the rats’ paws to withdraw was taken as the withdrawal threshold. Each animal underwent the MWT test 10 times. The cutoff value of the von Frey force was set at 15 g, and the results are shown in Table 1.

[0075] Table 1MWT measurement results

[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 negative control group); 2: P < 0.05 (compared with positive control group); 3: P < 0.05 (compared with Example 6 group)

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

[0079] Table 2 TWL measurement results

[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 negative control group); 2: P < 0.05 (compared with positive control group); 3: P < 0.05 (compared with Example 6 group)

[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 using the Sophora flavescens extract were significantly increased. Compared with the Example 6 group (single Sophora flavescens ethanol extract), the Example 2 group and the Example 5 group (containing Lactobacillus plantarum CGMCC 1.12935) were able to significantly improve the mechanical withdrawal threshold and the heat withdrawal latency, while the Example 3 group (containing Streptococcus thermophilus CGMCC 1.1481) and the Example 4 group (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 Sophora flavescens fermentation product, while other types of Lactobacillus plantarum or Streptococcus thermophilus alone cannot achieve such an effect; while the addition of Lactobacillus plantarum CGMCC 1.12935 and Streptococcus thermophilus CGMCC 1.1481 at the same time can significantly improve the mechanical withdrawal threshold and the heat withdrawal latency. The extract obtained by fermenting Sophora flavescens with 1.1481 had the strongest immune activity, and MWT and TWL increased significantly, approaching that of the sham operation group rats. It was considered that only the special fermentation of Streptococcus thermophilus CGMCC1.1481 could produce a synergistic effect with Lactobacillus plantarum CGMCC 1.12935, thereby changing the fermentation process of Lactobacillus plantarum CGMCC 1.12935 on Sophora flavescens, thereby improving the fermentation quality of Sophora flavescens fermented liquid.

[0083] Example 8

[0084] After the rats were tested, anesthesia was induced, saline was perfused into the heart, and the spinal cord region between the L4-L6 segments was obtained for subsequent experiments.

[0085] GFAP observation: The frozen sections were fixed. The sections were incubated with GFAP primary antibody (dilution: 1:200) at 4°C overnight, then the secondary antibody was added, incubated at 37°C for 30 min, counterstained and sealed, and observed under a microscope. The GFAP protein expression level was analyzed using Image J software, and the results are shown in Table 3.

[0086] Table 3Relative protein expression 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 negative control group); 2: P < 0.05 (compared with positive control group); 3: P < 0.05 (compared with Example 6 group)

[0089] Inflammatory factor determination: ELISA detection of inflammatory factor levels The spinal cord segments of the other 6 rats in each group were quickly frozen in liquid nitrogen, ground and made into homogenate. After centrifugation, the supernatant was taken and IL-1β and TNF-α were detected by ELISA kit, and the optical density value at 490nm was measured by microplate reader to analyze the average expression levels of IL-1β and TNF-α. The results are shown in Table 4.

[0090] Table 4 Inflammatory factor level determination (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 negative control group); 2: P < 0.05 (compared with positive control group); 3: P < 0.05 (compared with Example 6 group)

[0093] Compared with the sham operation group, the GFAP relative protein content in the spinal cord of the model group rats was the highest, and the IL-1β and TNF-α inflammatory factor levels were the highest. Compared with the Example 6 group (single Sophora flavescens alcohol extract), the inflammatory factor levels and GFAP of the Example 2 group and the Example 5 group (containing Lactobacillus plantarum CGMCC1.12935) decreased significantly, while the Example 3 group (containing Streptococcus thermophilus CGMCC 1.1481) and the Example 4 group (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 natural Sophora flavescens fermentation product; and the extract obtained by adding Lactobacillus plantarum CGMCC 1.12935 and Streptococcus thermophilus CGMCC 1.1481 to ferment Sophora flavescens has the strongest ability to reduce the activity of immune factors, and GFAP decreases the most, which is close to the sham operation group rats. The expression of GFAP protein was significantly upregulated, and the number of activated astrocytes increased. After adding Sophora flavescens for immune regulation, inflammatory factors decreased, the excessive activation of GFAP was inhibited, and the number of activated astrocytes was significantly reduced. It is considered that the fermentation of Sophora flavescens with Streptococcus thermophilus CGMCC 1.1481 and Lactobacillus plantarum CGMCC 1.12935 can produce a synergistic effect, which may produce more effective small molecules and greatly improve the fermentation quality of Sophora flavescens fermented liquid.

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

[0095] The description of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction 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 may be combined in any suitable manner in any one or more embodiments or examples.

[0096] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. An immunomodulator for inhibiting excessive activation of astrocytes, the preparation method of which is: (1) An appropriate amount of Sophora flavescens was dried and ground to pass through a 40-mesh sieve, 1 weight part of the ground Sophora flavescens was taken, 5 weight parts of distilled water was added, 1% of the seed solution of Lactobacillus plantarum CGMCC 1.12935 and 1% of the seed solution of Streptococcus thermophilus CGMCC 1.1481 were inoculated, and fermented at 37° C. for 24 hours; (2) Add 10 parts by weight of 75% ethanol and extract under reflux at 75°C once; add 10 parts by weight of 75% ethanol again and extract under reflux at 75°C once; combine the filtrates, concentrate the filtrates, filter through a 0.22 um filter membrane, evaporate to a thick consistency, and freeze-dry under vacuum at -80°C to obtain a yellow-white powder; store in a sealed container at 4°C.

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

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

4. The immunomodulator according to any one of claims 1 to 3, for use in treating neuropathic pain.