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      非瑟酮通過上調(diào)GPR35抑制嗎啡誘導的小鼠小膠質(zhì)細胞BV2活化和炎癥反應*

      2023-08-07 06:57:48關森王慧淼王莉萍孫艷斌
      中國病理生理雜志 2023年7期
      關鍵詞:嗎啡膠質(zhì)活化

      關森, 王慧淼, 王莉萍, 孫艷斌△

      非瑟酮通過上調(diào)GPR35抑制嗎啡誘導的小鼠小膠質(zhì)細胞BV2活化和炎癥反應*

      關森1, 王慧淼2, 王莉萍1, 孫艷斌1△

      (1承德市中心醫(yī)院麻醉科,河北 承德 067000;2承德市中心醫(yī)院檢驗科,河北 承德 067000)

      探討非瑟酮抑制嗎啡誘導的小鼠小膠質(zhì)細胞BV2活化和炎癥的機制。通過嗎啡干預BV2細胞構建細胞模型,將細胞隨機分為6組:對照組、嗎啡組、嗎啡+2 μmol/L非瑟酮組、嗎啡+4 μmol/L非瑟酮組、嗎啡+8 μmol/L非瑟酮組和嗎啡+10 μmol/L米諾環(huán)素組,實驗重復3次。CCK-8法檢測細胞活力;ELISA試劑盒檢測細胞上清液中炎癥因子水平;Western blot檢測炎癥相關蛋白及BV2細胞活化標志物表達水平;RT-qPCR和Western blot檢測G蛋白偶聯(lián)受體35(GPR35)的表達;BV2細胞轉染干擾質(zhì)粒驗證非瑟酮是否通過調(diào)控GPR35抑制嗎啡誘導的BV2細胞活化和炎癥反應。與對照組相比,不同劑量非瑟酮單獨處理后BV2細胞活力均無顯著差異;嗎啡組細胞活力升高,炎癥因子[腫瘤壞死因子α(TNF-α)、白細胞介素1β(IL-1β)和IL-6]、環(huán)加氧酶2(Cox-2)和誘導型一氧化氮合酶(iNOS)蛋白表達增加,BV2細胞活化標志物離子鈣結合接頭分子1(Iba-1)和CD11b表達上調(diào)(<0.05)。與嗎啡組相比,嗎啡+非瑟酮組和嗎啡+米諾環(huán)素組細胞活力降低,炎癥因子(TNF-α、IL-1β和IL-6)、Cox-2和iNOS蛋白表達減少,BV2細胞活化標志物Iba-1和CD11b表達下調(diào)(<0.05)。RT-qPCR和Western blot結果顯示,非瑟酮劑量依賴性地增加嗎啡誘導的BV2細胞中GPR35的表達(<0.05)。干擾逆轉非瑟酮對嗎啡誘導的BV2細胞活化和炎癥反應的抑制作用。非瑟酮可能通過上調(diào)GPR35抑制嗎啡誘導的小鼠小膠質(zhì)細胞BV2活化和炎癥。

      非瑟酮;G蛋白偶聯(lián)受體35;小膠質(zhì)細胞;炎癥

      嗎啡(morphine)等阿片類藥物是治療慢性疼痛的強效鎮(zhèn)痛藥物,廣泛應用于臨床[1]。但藥物耐受性、痛覺過敏等副作用阻礙了這類藥物在臨床上的長期應用[2]。因此,迫切需要能夠增強鎮(zhèn)痛效果同時降低耐受性和副作用的治療策略,以提高患者的生活質(zhì)量。

      盡管阿片類藥物耐受的確切機制很復雜且目前尚未有很好的解釋,但有證據(jù)表明,小膠質(zhì)細胞的激活和隨之而來的促炎細胞因子產(chǎn)生起著關鍵的致病作用[3-4]?;罨男∧z質(zhì)細胞經(jīng)歷從靜止到巨噬細胞樣的顯著形態(tài)變化,伴隨著細胞表面標志物的表達增加,例如CD11b、CD14、主要組織相容性復合物分子、趨化因子受體和其他幾種標志物,產(chǎn)生大量促炎細胞因子,包括白細胞介素1β(interleukin-1β, IL-1β)、腫瘤壞死因子α(tumor necrosis factor-α, TNF-α)和IL-6,它們可以增強背角神經(jīng)元的反應性,誘導中樞敏化,降低嗎啡的鎮(zhèn)痛作用[5-6]。盡管小膠質(zhì)細胞活化的抑制可能是改善嗎啡鎮(zhèn)痛作用的潛在治療策略[7],具體調(diào)控機制尚不清楚。

      非瑟酮(fisetin)是一種廣泛存在于蔬菜和水果中的生物活性黃烷醇[8],是中藥黃檗的有效成分。越來越多的證據(jù)表明非瑟酮具有多種藥理作用,包括抗腫瘤、抗血管生成、抗衰老和神經(jīng)保護特性[9-12]。非瑟酮在腦小膠質(zhì)細胞中具有很強的抗炎活性,可能是治療神經(jīng)炎性疾病的潛在藥物[13]。非瑟酮通過抑制小膠質(zhì)細胞中IL-1受體1、磷酸化核因子κB、TNF-α和誘導型一氧化氮合酶(inducible nitric oxide synthase, iNOS)表達,抑制神經(jīng)炎癥,緩解膿毒癥相關性腦病大鼠的認知功能障礙[14]。在退行性神經(jīng)疾病模型中,非瑟酮顯著減輕炎癥引起的運動和協(xié)調(diào)功能缺陷,抑制小膠質(zhì)細胞活化[15]。另外,慢性非瑟酮治療可延遲或糾正1型糖尿病小鼠的神經(jīng)性痛覺過敏和異常性疼痛[16],在神經(jīng)性疼痛小鼠模型中發(fā)揮抗痛覺過敏作用[17]??紤]到非瑟酮的這些藥理活性,推測非瑟酮可能對嗎啡誘導的小膠質(zhì)細胞活化和神經(jīng)炎癥具有抑制作用。因此,本項工作探討非瑟酮對嗎啡誘導的小鼠小膠質(zhì)細胞BV2活化和炎癥的作用及其機制。

      材料和方法

      1 細胞與試劑

      小鼠小膠質(zhì)細胞系BV2購自北京北納培養(yǎng)物保藏中心;非瑟酮和米諾環(huán)素(minocycline)購自Sigma;鹽酸嗎啡購自東北醫(yī)藥集團公司沈陽第一制藥廠;DMEM培養(yǎng)液、胎牛血清(fetal bovine serum, FBS)、青霉素和鏈霉素均購自Gibco;Trizol RNA分離試劑、逆轉錄試劑盒及SYBR Green試劑盒均購自Thermo Fisher Scientific;CCK-8試劑盒購自Dojindo Molecular Technologies;ELISA試劑盒購自上海酶聯(lián)生物技術有限公司;環(huán)加氧酶2(cyclooxygenase-2, Cox-2)抗體(ab179800)、iNOS抗體(ab178945)、離子鈣結合接頭分子1(ionized calcium-binding adapter molecule-1, Iba-1)抗體(ab178846)、CD11b抗體(ab133357)、G蛋白偶聯(lián)受體35(G protein-coupled receptor 35, GPR35)抗體(ab76217)、GAPDH抗體(ab9485)及辣根過氧化物酶標記的羊抗兔IgG均購自Abcam。

      2 細胞培養(yǎng)、造模及轉染

      BV2細胞用含10% FBS、100 U/mL青霉素、100 mg/L鏈霉素和2 mmol/L谷氨酰胺的DMEM培養(yǎng)液在濕潤的5% CO2和95%空氣的37 ℃環(huán)境中培養(yǎng)。將細胞隨機分為對照組、嗎啡組、嗎啡+2 μmol/L非瑟酮組、嗎啡+4 μmol/L非瑟酮組、嗎啡+8 μmol/L非瑟酮組和嗎啡+10 μmol/L米諾環(huán)素組,對于嗎啡誘導和藥物治療,將BV2細胞接種在6孔板中(每孔1×105細胞)過夜。用非瑟酮(2、4和8 μmol/L)處理細胞2 h,用小膠質(zhì)細胞活化抑制劑米諾環(huán)素(10 μmol/L)作為陽性對照,然后200 μmol/L嗎啡誘導細胞6 h[18]。

      將由上?;蛑扑幱邢薰咎峁┑母蓴_質(zhì)粒及陰性對照按LipofectamineTM3000說明書步驟轉染到BV2細胞中,并通過RT-qPCR和Western blot檢測轉染效率。

      3 方法

      3.1CCK-8實驗將BV2細胞接種在96孔板中(每孔5×103個細胞)中,37 ℃培養(yǎng)過夜。按分組對細胞進行相應處理后,每孔加入10 μL CCK-8試劑,在細胞培養(yǎng)箱中培養(yǎng)2 h。使用酶標儀檢測450 nm處吸光度,計算各組細胞活力。實驗重復3次。

      3.2RT-qPCR實驗使用Trizol試劑從BV2細胞中提取總RNA,使用逆轉錄試劑盒合成第一鏈cDNA,進一步通過SYBR Green進行RT-qPCR。將合成的cDNA用SYBR Premix Taq在ABI 7500實時PCR系統(tǒng)溫控器上進行定量PCR。擴增方法:95 ℃ 10 min;95 ℃ 15 s, 60 ℃ 1 min,共40個循環(huán)。以GAPDH為內(nèi)參照進行歸一化處理,并用2-ΔΔCt法計算目的基因的相對表達水平。引物序列見表1。

      表1 RT-qPCR引物序列

      3.3ELISA實驗將處理后的BV2細胞在PBS中勻漿,離心后采用ELISA試劑盒檢測細胞上清液中TNF-α、IL-1β和IL-6水平。

      3.4Western blot實驗收集每組細胞后,用RIPA裂解液提取BV2細胞中的總蛋白。根據(jù)BCA蛋白濃度測定試劑盒說明書測定提取的蛋白濃度。取30 μg總蛋白上樣進行SDS-PAGE后,通過轉膜儀將蛋白轉移到PVDF膜上。5%脫脂奶粉室溫封閉1 h后,4 ℃ Ⅰ抗孵育過夜。用TBST洗膜3次后,室溫下加入辣根過氧化物酶標記的Ⅱ抗孵育2 h TBST洗膜3次,每次10 min。按照ECL化學發(fā)光試劑盒的說明顯色條帶,然后用ImageJ軟件分析條帶的灰度值。

      4 統(tǒng)計學處理

      所有實驗均進行3次平行實驗,數(shù)據(jù)表示為均數(shù)±標準差(meanSD),并用GraphPad Prism 8.0進行統(tǒng)計學分析,組間均數(shù)比較采用單因素方差分析,以<0.05為差異有統(tǒng)計學意義。

      結果

      1 非瑟酮對嗎啡誘導的BV2細胞活力的作用

      利用CCK-8法檢測BV2細胞活力,如圖1A所示,2、4和8 μmol/L非瑟酮對BV2細胞不具有細胞毒性(>0.05),因此選擇2、4和8 μmol/L非瑟酮進行下一步研究。如圖1B所示,與對照組相比,嗎啡組細胞活力升高(<0.05),而4和8 μmol/L非瑟酮及10 μmol/L米諾環(huán)素處理均能夠降低BV2細胞活力(<0.05或<0.01)。

      Figure 1. Effect of fisetin on the viability of BV2 cells induced by morphine. A: CCK-8 assay was used to detect the cytotoxicity of fisetin; B: CCK-8 assay was used to detect morphine-induced BV2 cell viability. Mean±SD. n=3. **P<0.01 vs control group; #P<0.05, ##P<0.01 vs morphine group.

      2 非瑟酮抑制嗎啡誘導的BV2細胞炎癥反應

      ELISA試劑盒和Western blot被用來檢測BV2細胞炎癥水平。與對照組相比,嗎啡組細胞上清液中炎癥因子TNF-α、IL-1β和IL-6水平顯著升高,炎癥相關蛋白Cox-2和iNOS表達顯著增加(<0.05);與嗎啡組相比,不同劑量非瑟酮及米諾環(huán)素處理后細胞上清液中炎癥因子TNF-α、IL-1β和IL-6水平顯著降低,炎癥相關蛋白Cox-2和iNOS表達顯著減少(<0.05),見圖2。

      Figure 2. Effect of fisetin on morphine-induced inflammatory response in BV2 cells. A: the levels of TNF-α, IL-1β and IL-6 in BV2 cell supernatant were detected by ELISA kit; B: the expression levels of inflammation-related proteins Cox-2 and iNOS in BV2 cells were detected by Western blot. Mean±SD. n=3. **P<0.01 vs control group; ##P<0.01 vs morphine group.

      3 非瑟酮抑制嗎啡誘導的BV2細胞活化

      采用Western blot檢測BV2細胞活化標志蛋白Iba-1和CD11b的表達水平。與對照組相比,嗎啡組細胞中Iba-1和CD11b表達顯著增加(<0.01);與嗎啡組相比,不同劑量非瑟酮及米諾環(huán)素處理后細胞中Iba-1和CD11b表達顯著減少(<0.01),見圖3。

      Figure 3. Effect of fisetin on morphine-induced activation of BV2 cells. The expression levels of Iba-1 and CD11b in BV2 cells were detected by Western blot. Mean±SD. n=3. **P<0.01 vs control group; ##P<0.01 vs morphine group.

      4 非瑟酮上調(diào)嗎啡誘導的BV2細胞中GPR35表達

      RT-qPCR和Western blot結果顯示,與對照組相比,嗎啡組細胞中GPR35的mRNA水平顯著降低,蛋白表達顯著減少(<0.01);與嗎啡組相比,不同劑量非瑟酮及米諾環(huán)素處理后細胞中GPR35的mRNA水平顯著升高,蛋白表達顯著增多(<0.01),見圖4。下一步研究選擇8 μmol/L非瑟酮進行。

      Figure 4. fisetin regulates the expression of GPR35 induced by morphine in BV2 cells. A: RT-qPCR was used to detect the expression level of GPR35 mRNA in BV2 cells; B: Western blot was used to detect the protein expression level of GPR35 in BV2 cells. Mean±SD. n=3. **P<0.01 vs control group; ##P<0.01 vs morphine group.

      5 非瑟酮通過上調(diào)GPR35抑制嗎啡誘導的BV2細胞炎癥水平

      對BV2細胞轉染干擾質(zhì)粒,再采用RT-qPCR和Western blot檢測干擾效率。如圖5A、B所示,干擾質(zhì)粒構建成功,并選擇sh--1進行后續(xù)研究。ELISA和Western blot結果顯示,與嗎啡組相比,嗎啡+非瑟酮組BV2細胞上清液中炎癥因子TNF-α、IL-1β和IL-6水平顯著降低,炎癥相關蛋白Cox-2和iNOS表達顯著減少(<0.01);與嗎啡+非瑟酮組相比,嗎啡+非瑟酮+sh-NC組BV2細胞炎癥水平無顯著差異,嗎啡+非瑟酮+sh-GPR35組BV2細胞上清液中炎癥因子TNF-α、IL-1β和IL-6水平顯著升高,炎癥相關蛋白Cox-2和iNOS表達顯著增多(<0.01),見圖5C、D。

      Figure 5. Effect of fisetin on morphine-induced inflammation level in BV2 cells by regulating GPR35. A: RT-qPCR was used to detect the mRNA level of GPR35; B: Western blot was used to detect the protein level of GPR35; C: ELISA kit was used to detect the levels of inflammatory factors TNF-α, IL-1β and IL-6 in the cell supernatant; D: Western blot was used to detect the expression levels of inflammation-related proteins Cox-2 and iNOS in BV2 cells. Mean±SD. n=3. **P<0.01 vs control group; ##P<0.01 vs morphine group.

      6 非瑟酮通過上調(diào)GPR35抑制嗎啡誘導的BV2細胞活化

      Western blot結果顯示,與嗎啡組相比,嗎啡+非瑟酮組BV2細胞中Iba-1和CD11b表達水平顯著降低(<0.05);與嗎啡+非瑟酮組相比,嗎啡+非瑟酮+sh-NC組BV2細胞活化標志蛋白表達水平無顯著差異,嗎啡+非瑟酮+sh-GPR35組BV2細胞中Iba-1和CD11b表達水平顯著升高(<0.05),見圖6。

      Figure 6. Effect of fisetin on morphine-induced activation of BV2 cells by regulating GPR35. The expression levels of Iba-1 and CD11b in BV2 cells were detected by Western blot. Mean±SD. n=3. **P<0.01 vs control group; ##P<0.01 vs morphine group.

      討論

      本研究證明了黃酮類化合物非瑟酮對嗎啡誘導的小膠質(zhì)細胞活化和炎癥反應具有抑制作用。鑒于小膠質(zhì)細胞活化和神經(jīng)炎癥已被廣泛認為是嗎啡耐受的重要因素,提示利用藥物抑制小膠質(zhì)細胞活化能夠緩解嗎啡耐受,從而增強嗎啡的鎮(zhèn)痛作用。但是常見的小膠質(zhì)細胞活化抑制劑米諾環(huán)素是一種廣譜抗生素,長期使用可能會造成多種不良反應。因此,找到尋找到一種安全有效的抑制小膠質(zhì)細胞活化的藥物具有重要的意義。非瑟酮作為中藥單體,在臨床上已經(jīng)有廣泛的應用,并且具有相對較高的安全性。本研究表明,非瑟酮能夠在體外劑量依賴性地抑制嗎啡誘導的BV2細胞中炎癥因子和活化標志物的表達,提示非瑟酮有潛力成為這種安全有效的小膠質(zhì)細胞活化抑制劑。

      為了探討非瑟酮抑制嗎啡誘導的BV2細胞活化和炎癥反應的機制,利用SwissTargetPrediction網(wǎng)站對非瑟酮可能的作用靶點進行預測,顯示非瑟酮能夠靶向調(diào)控GPR35。G蛋白偶聯(lián)受體是最大的細胞表面受體家族,在調(diào)節(jié)許多重要的生理功能中起關鍵作用。激素、光、脂質(zhì)、氣味劑和神經(jīng)遞質(zhì)等興奮劑可激活這些受體[19],這些受體被激活后可調(diào)節(jié)多種細胞內(nèi)信號通路,從而參與心血管疾病病、腫瘤、免疫和感染性疾病等的發(fā)生發(fā)展。GPR35是一種視紫紅質(zhì)樣的A類G蛋白偶聯(lián)受體,以區(qū)域特異性方式在中樞和周圍神經(jīng)系統(tǒng)中表達,例如小鼠延髓、海馬、脊髓和背根神經(jīng)節(jié)[20]。本研究結果表明,非瑟酮處理可逆轉嗎啡誘導的BV2細胞中GPR35 mRNA和蛋白表達的下調(diào)。有文獻報道,GPR35在腦缺血[21]、炎性腸?。?2]、子宮內(nèi)膜炎[23]多種疾病中發(fā)揮抗炎作用。通過實驗我們觀察到,干擾能夠逆轉非瑟酮對嗎啡誘導的BV2細胞炎癥反應的抑制作用。GPR35激動劑扎普司特在大鼠神經(jīng)性疼痛模型中減輕了疼痛癥狀并增強了嗎啡和丁丙諾啡的鎮(zhèn)痛特性[24],提示GPR35在嗎啡鎮(zhèn)痛中的有益作用。而本項工作也揭示了干擾能夠逆轉非瑟酮對嗎啡誘導的BV2細胞活化的抑制作用。因此,激活GPR35能夠抑制嗎啡誘導的BV2細胞活化,緩解嗎啡耐受,增強嗎啡的鎮(zhèn)痛作用。

      綜上所述,本研究顯示非瑟酮通過靶向上調(diào)GPR35抑制嗎啡誘導的小鼠BV2細胞活化和炎癥反應,從而為緩解嗎啡耐受提供了參考資料。

      [1] Volkow ND, McLellan AT. Opioid abuse in chronic pain-misconceptions and mitigation strategies[J]. N Engl J Med, 2016, 374(13):1253-1263.

      [2] Horvath RJ, DeLeo JA. Morphine enhances microglial migration through modulation of P2X4receptor signaling[J]. J Neurosci, 2009, 29(4):998-1005.

      [3] Ferrini F, Trang T, Mattioli TA, et al. Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl? homeostasis[J]. Nat Neurosci, 2013, 16(2):183-192.

      [4] Vacca V, Marinelli S, Luvisetto S, et al. Botulinum toxin A increases analgesic effects of morphine, counters development of morphine tolerance and modulates glia activation and μ opioid receptor expression in neuropathic mice[J]. Brain Behav Immun, 2013, 32:40-50.

      [5] Berta T, Liu YC, Xu ZZ, et al. Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1β and ERK signaling in the spinal cord of mice[J]. Neuroscience, 2013, 247:376-385.

      [6] Taves S, Berta T, Chen G, et al. Microglia and spinal cord synaptic plasticity in persistent pain[J]. Neural Plast, 2013, 2013:753656.

      [7] Pan Y, Sun X, Jiang L, et al. Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation[J]. J Neuroinflammation, 2016, 13(1):294.

      [8] Trzeciak A, Pietropaoli AP, Kim M. Biomarkers and associated immune mechanisms for early detection and therapeutic management of sepsis[J]. Immune Netw, 2020, 20(3):e23.

      [9] Zheng W, Feng Z, You S, et al. Fisetin inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes through activating SIRT1 and attenuates the progression of osteoarthritis in mice[J]. Int Immunopharmacol, 2017, 45:135-147.

      [10] Lorthongpanich C, Charoenwongpaiboon T, Supakun P, et al. Fisetin inhibits osteogenic differentiation of mesenchymal stem cells via the inhibition of YAP[J]. Antioxidants (Basel), 2021, 10(6):879.

      [11] Molagoda IMN, Jayasingha JACC, Choi YH, et al. Fisetin inhibits lipopolysaccharide-induced inflammatory response by activating β-catenin, leading to a decrease in endotoxic shock[J]. Sci Rep, 2021, 11(1):8377.

      [12] Kirkland JL, Tchkonia T. Senolytic drugs: from discovery to translation[J]. J Intern Med, 2020, 288(5):518-536.

      [13] ZhengLT, Ock J, Kwon BM, etal. Suppressive effects of flavonoid fisetin on lipopolysaccharide-induced microglial activation and neurotoxicity[J]. Int Immunopharmacol, 2008, 8(3):484-494.

      [14] Ding H, Li Y, Chen S, et al. Fisetin ameliorates cognitive impairment by activating mitophagy and suppressing neuroinflammation in rats with sepsis-associated encephalopathy[J]. CNS Neurosci Ther, 2022, 28(2):247-258.

      [15] Chuang JY, Chang PC, Shen YC, et al. Regulatory effects of fisetin on microglial activation[J]. Molecules, 2014, 19(7):8820-8839.

      [16] Zhao X, Li XL, Liu X, et al. Antinociceptive effects of fisetin against diabetic neuropathic pain in mice: engagement of antioxidant mechanisms and spinal GABAAreceptors[J]. Pharmacol Res, 2015, 102:286-297.

      [17] Zhao X, Wang C, Cui WG, et al. Fisetin exerts antihyperalgesic effect in a mouse model of neuropathic pain: engagement of spinal serotonergic system[J]. Sci Rep, 2015, 5(1):9043.

      [18] Guan S, Jin T, Han S, et al. Dihydroartemisinin alleviates morphine-induced neuroinflammation in BV-2 cells[J]. Bioengineered, 2021, 12(2):9401-9410.

      [19] Khan MZ, He L. Neuro-psychopharmacological perspective of orphan receptors of rhodopsin (class A) family of G protein-coupled receptors[J]. Psychopharmacology (Berl), 2017, 234(8):1181-1207.

      [20] Berlinguer-Palmini R, Masi A, Narducci R, et al. GPR35 activation reduces Ca2+transients and contributes to the kynurenic acid-dependent reduction of synaptic activity at CA3-CA1 synapses[J]. PLoS One, 2013, 8(11):e82180.

      [21] Sharmin O, Abir AH, Potol A, et al. Activation of GPR35 protects against cerebral ischemia by recruiting monocyte-derived macrophages[J]. Sci Rep, 2020, 10(1):9400.

      [22] Farooq SM, Hou Y, Li H, et al. Disruption of GPR35 exacerbates dextran sulfate sodium-induced colitis in mice[J]. Dig Dis Sci, 2018, 63(11):2910-2922.

      [23] Wang Y, Liu Z, Shen P, et al. Kynurenic acid ameliorates lipopolysaccharide-induced endometritis by regulating the GRP35/NF-κB signaling pathway[J]. Toxicol Appl Pharmacol, 2022, 438:115907.

      [24] Rojewska E, Piotrowska A, Jurga A, et al. Zaprinast diminished pain and enhanced opioid analgesia in a rat neuropathic pain model[J]. Eur J Pharmacol, 2018, 839:21-32.

      Fisetin inhibits activation and inflammatory response of mouse microglia BV2 cells induced by morphine via up-regulating GPR35

      GUAN Sen1, WANG Huimiao2, WANG Liping1, SUN Yanbin1△

      (1,,067000,;2,,067000,)

      To investigate the impacts of fisetin on morphine-induced activation and inflammation of mouse microglia BV2 cells, and to discuss its mechanism.Initially, morphine was applied for the induction of BV2 cells. The cells were divided into 6 groups: control group, morphine group, morphine+2 μmol/L fisetin group, morphine+4 μmol/L fisetin group, morphine+8 μmol/L fisetin group and morphine+10 μmol/L minocycline group. The cell viability was detected by CCK-8 assay, and ELISA kits were used to detect the levels of inflammatory factors in cell supernatants. The expression levels of inflammation-related proteins and BV2 cell activation markers were measured by Western blot. The mRNA and protein expression levels of G protein-coupled receptor 35 (GPR35) were evaluated by RT-qPCR and Western blot. TheBV2 cells were transfected withinterfering plasmid to verify whether fisetin inhibited morphine-induced BV2 cell activation and inflammatory response by regulating GPR35.Compared with control group, fisetin had no significant effect on the viability of BV2 cells. Morphine increased the viability of BV2 cells, and elevated the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, cyclooxygenase-2 (Cox-2), inducible nitric oxide synthase (iNOS), ionized calcium-binding adapter molecule-1 (Iba-1) and CD11b (<0.05). The BV2 cell viability and the expression levels of TNF-α, IL-1β, IL-6, Cox-2, iNOS, Iba-1 and CD11b in morphine-induced BV2 cells were reduced after fisetin or minocycline treatment (<0.05). Fisetin increased the mRNA and protein expression levels of GPR35 in morphine-induced BV2 cells in a dose-dependent manner (<0.05). The rescue experiments confirmed thatknockdown reversed the inhibitory effects of fisetin on morphine-induced BV2 cell activation and inflammatory response.Fisetin inhibits morphine-induced BV2 cell activation and inflammation by up-regulating GPR35.

      fisetin; G protein-coupled receptor 35 ; microglia; inflammation

      1000-4718(2023)07-1218-07

      2022-07-21

      2023-04-03

      18730488200; E-mail: sun0403@163.com

      R741; R363.2

      A

      10.3969/j.issn.1000-4718.2023.07.008

      [基金項目]承德市科學技術研究與發(fā)展計劃項目(No. 202102A009)

      (責任編輯:宋延君,李淑媛)

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